Fc-region variants with modified fcrn- and protein a-binding properties

ABSTRACT

Herein is reported a heterodimeric polypeptide comprising a first polypeptide comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, and a second polypeptide comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V (hole-chain) and the second polypeptide comprises the mutations S354C and T366W (knob-chain), and wherein the first polypeptide (hole-chain) comprises the mutations i) I253A or I253G, and ii) L314A or L314G or L314D, and wherein the first polypeptide and the second polypeptide are connected by one or more disulfide bridges, and wherein the CH3-domain of the first polypeptide and the CH3-domain of the second polypeptide both bind or both do not bind to protein A (numbering according to the Kabat EU index).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/586,686, filed May 4, 2017 which is a continuation of InternationalApplication Number PCT/EP2015/075657, filed Nov. 4, 2015, which claimsbenefit under 35 U.S.C. § 119 to European Application Number 14192054.6,filed Nov. 6, 2014 which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequence Listing filed concurrently herewith. The material inthe electronic Sequence Listing is submitted as a text file entitledSequence_Listing.txt created on Dec. 15, 2020 which has a file size of58.0 KB, and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

Herein are reported IgG Fc-regions that have been modified with respectto Fc-receptor as well as protein A binding.

BACKGROUND OF THE INVENTION

The demand for cost efficient production processes has led to thenecessity of optimization of the downstream purification, including oneor more affinity chromatography steps. Larger volumes to be processedand harder requirements for the cleaning-in-place (CIP) protocols aresome of the features that need to be solved (Hober, S., J. Chrom. B. 848(2007) 40-47).

The purification of monoclonal antibodies by means of selectiveFc-region affinity ligands is the most promising methodology for thelarge-scale production of therapeutic monoclonal antibodies. In fact,this procedure does not require establishing any interaction with theantigen specific part of the antibody, i.e. the Fab domain, which is,thus, left intact and can retain its properties (see Salvalaglio, M., etal., J. Chrom. A 1216 (2009) 8678-8686).

Due to its selectiveness, an affinity-purification step is employedearly in the purification chain and thereby the number of successiveunit operations can be reduced (see Hober supra; MacLennan, J.,Biotechnol. 13 (1995) 1180; Harakas, N. K., Bioprocess Technol. 18(1994) 259).

The ligands most adopted to bind selectively IgG are Staphylococcalprotein A and protein G, which are able to establish highly selectiveinteractions with the Fc-region of most IgGs in a region known as“consensus binding site” (CBS) (DeLano, W. L., et al., Science 287(2000) 1279), which is located at the hinge region between the CH2 andCH3 domains of the Fc-region.

Staphylococcal protein A (SPA) is a cell wall associated protein domainexposed on the surface of the Gram-positive bacterium Staphylococcusaureus. SPA has high affinity to IgG from various species, for instancehuman, rabbit and guinea pig IgG but only weak interaction with bovineand mouse IgG (see the following Table) (see Hober supra; Duhamel, R.C., et al., J. Immunol. Methods 31 (1979) 211; Björk, L. and Kronvall,G., Immunol. J. 133 (1984) 969; Richman, D. D., et al., J. Immunol. 128(1982) 2300; Amersham Pharmacia Biotech, Handbook, Antibody Purification(2000)).

species subclass protein A binding human IgG1 ++ IgG2 ++ IgG3 −− IgG4 ++IgA variable IgD − IgM variable rabbit no distinction ++ guinea pig IgG1++ IgG2 ++ bovine + mouse IgG1 + IgG2a ++ IgG2b + IgG3 + IgM variablechicken IgY − ++: strong binding/ +: medium binding/ −: weak or nointeraction

The heavy chain hinge-region between the CH2 and CH3 domains of IgG isable to bind several proteins beyond protein A, such as the neonatal Fcreceptor (FcRn) (see DeLano and Salvalaglio supra).

The SPA CBS comprehends a hydrophobic pocket on the surface of theantibody. The residues composing the IgG CBS are Ile 253, Ser 254, Met252, Met 423, Tyr 326, His 435, Asn 434, His 433, Arg 255, and Glu 380(numbering of the IgG heavy chain residues according to the Kabat EUindex numbering system). The charged amino acids (Arg 255, Glu 380) areplaced around a hydrophobic knob formed by Ile 253 and Ser 254. This(can) result in the establishment of polar and hydrophilic interactions(see Salvalaglio supra).

In general, the protein A-IgG interaction can be described using twomain binding sites: the first is positioned in the heavy chain CH2domain and is characterized by hydrophobic interactions between Phe 132,Leu 136, Ile 150 (of protein A) and the IgG hydrophobic knob constitutedby Ile 253 and Ser 254, and by one electrostatic interaction between Lys154 (protein A) and Thr 256 (IgG). The second site is located in theheavy chain CH3 domain and is dominated by electrostatic interactionsbetween Gln 129 and Tyr 133 (protein A) and His 433, Asn 434, and His435 (IgG) (see Salvalaglio supra).

Lindhofer, H., et al. (J. Immunol. 155 (1995) 219-225) reportpreferential species-restricted heavy/light chain pairing in rat/mousequadromas.

Jedenberg, L., et al. (J. Immunol. Meth. 201 (1997) 25-34) reported thatSPA-binding analyses of two Fc variants (Fc13 and Fc31, each containingan isotypic dipeptide substitution from the respective other isotype)showed that Fc1 and Fc31 interact with SPA, while Fc3 and Fc13 lackdetectable SPA binding. The rendered SPA binding of the Fc-regionvariant Fc31 is concluded to result from the introduced dipeptidesubstitution R435H and F436Y.

Today the focus with respect to therapeutic monoclonal antibodies is onthe generation and use of bispecific or even multispecific antibodiesspecifically binding to two or more targets (antigens).

The basic challenge in generating multispecific heterodimeric IgGantibodies from four antibody chains (two different heavy chains and twodifferent light chains) in one expression cell line is the so-calledchain association issue (see Klein, C., et al., mAbs 4 (2012) 653-663).The required use of different chains as the left and the right arm ofthe multispecific antibody leads to antibody mixtures upon expression inone cell: the two heavy chains are able to (theoretically) associate infour different combinations (two thereof are identical), and each ofthose can associate in a stochastic manner with the light chains,resulting in 2⁴ (=a total of 16) theoretically possible chaincombinations. Of the 16 theoretically possible combinations ten can befound of which only one corresponds to the desired functional bispecificantibody (De Lau, W. B., et al., J. Immunol. 146 (1991) 906-914). Thedifficulties in isolating this desired bispecific antibody out ofcomplex mixtures and the inherent poor yield of 12.5% at a theoreticalmaximum make the production of a bispecific antibody in one expressioncell line extremely challenging.

To overcome the chain association issue and enforce the correctassociation of the two different heavy chains, in the late 1990s Carteret al. from Genentech invented an approach termed “knobs-into-holes”(KiH) (see Carter, P., J. Immunol. Meth. 248 (2001) 7-15; Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681; Zhu, Z., et al., Prot.Sci. 6 (1997) 781-788; Ridgway, J. B., et al., Prot. Eng. 9 (1996)617-621; Atwell, S., et al., J. Mol. Biol. 270(1997) 26-35; and U.S.Pat. No. 7,183,076). Basically, the concept relies on modifications ofthe interface between the two CH3 domains of the two heavy chains of anantibody where most interactions occur. A bulky residue is introducedinto the CH3 domain of one antibody heavy chain and acts similarly to akey (“knob”). In the other heavy chain, a “hole” is formed that is ableto accommodate this bulky residue, mimicking a lock. The resultingheterodimeric Fc-region can be further stabilized by theintroduction/formation of artificial disulfide bridges. Notably, all KiHmutations are buried within the CH3 domains and not “visible” to theimmune system. In addition, properties of antibodies with KiH mutationssuch as (thermal) stability, FcγR binding and effector functions (e.g.,ADCC, FcRn binding) and pharmacokinetic (PK) behavior are not affected.

Correct heavy chain association with heterodimerization yields above 97%can be achieved by introducing six mutations: S354C, T366W in the “knob”heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain(see Carter supra; numbering of the residues according to the Kabat EUindex numbering system). While hole-hole homodimers may occur, knob-knobhomodimers typically are not observed. Hole-hole dimers can either bedepleted by selective purification procedures or by procedures asoutlined below.

While the issue of random heavy chain association has been addressed,also correct light chain association has to be ensured. Similar to theKiH CH3 domain approach, efforts have been undertaken to investigateasymmetric light chain-heavy chain interactions that might ultimatelylead to full bispecific IgGs.

Roche recently developed the CrossMab approach as a possibility toenforce correct light chain pairing in bispecific heterodimeric IgGantibodies when combining it with the KiH technology (see Klein supra;Schaefer. W., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192;Cain, C., SciBX 4 (2011) 1-4). This allows the generation of bispecificor even multispecific antibodies in a generic fashion. In this format,one arm of the intended bispecific antibody is left untouched. In thesecond arm, the whole Fab region, or the VH-VL domains or the CH1-CLdomains are exchanged by domain crossover between the heavy and lightchain. As a consequence, the newly formed “crossed” light chain does notassociate with the (normal, i.e. not-crossed) heavy chain Fab region ofthe other arm of the bispecific antibody any longer. Thus, the correct“light chain” association can be enforced by this minimal change indomain arrangement (see Schaefer supra).

Zhu et al. introduced several sterically complementary mutations, aswell as disulfide bridges, in the two VL/VH interfaces of diabodyvariants. When the mutations VL Y87A/F98M and VH V37F/L45W wereintroduced into the anti-p185HER2 VL/VH interface, a heterodimericdiabody was recovered with >90% yield while maintaining overall yieldand affinity compared with the parental diabody (see Zhu supra).

Researchers from Chugai have similarly designed bispecific diabodies byintroduction of mutations into the VH-VL interfaces (mainly conversionof Q39 in VH and Q38 in VL to charged residues) to foster correct lightchain association (WO 2006/106905; Igawa, T., et al., Prot. Eng. Des.Sel. 23 (2010) 667-677).

In WO2011097603 a common light chain mouse is reported.

In WO2010151792 a bispecific antibody format providing ease of isolationis provided, comprising immunoglobulin heavy chain variable domains thatare differentially modified, i.e. heterodimeric, in the CH3 domain,wherein the differential modifications are non-immunogenic orsubstantially non-immunogenic with respect to the CH3 modifications, andat least one of the modifications results in a differential affinity forthe bispecific antibody for an affinity reagent such as protein A, andthe bispecific antibody is isolable from a disrupted cell, from medium,or from a mixture of antibodies based on its affinity for protein A.

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvage IgGfrom the lysosomal degradation pathway, resulting in reduced clearanceand increased half-life. It is a heterodimeric protein consisting of twopolypeptides: a 50 kDa class I major histocompatibility complex-likeprotein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds withhigh affinity to the CH2-CH3 portion of the Fc-region of an antibody ofthe class IgG. The interaction between an antibody of the class IgG andthe FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgGantibody molecule can interact with two FcRn molecules via its two heavychain Fc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol.Biol. 230 (1993) 1077-1083).

Thus, an IgGs in vitro FcRn binding properties/characteristics areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class different amino acid residues of the heavy chain CH2- andCH3-domain are participating.

Different mutations that influence the FcRn binding and therewith thehalf-live in the blood circulation are known. Fc-region residuescritical to the mouse Fc-region-mouse FcRn interaction have beenidentified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., etal. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433, N434,and H435 (numbering according to Kabat EU index numbering system) areinvolved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26(1996) 2533-2536; Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Kim, J. K., et al., Eur. J. Immunol. 24 (1994) 542-548). Residues 1253,H310, and H435 were found to be critical for the interaction of humanFc-region with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29(1999) 2819-2885).

Methods to increase Fc-region (and likewise IgG) binding to FcRn havebeen performed by mutating various amino acid residues in the Fc-region:Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433,and Asn 434 (see Kuo, T. T., et al., J. Clin. Immunol. 30 (2010)777-789; Ropeenian, D. C., et al., Nat. Rev. Immunol. 7 (2007) 715-725).

The combination of the mutations M252Y, S254T, T256E has been describedby Dall'Acqua et al. to improve FcRn binding by protein-proteininteraction studies (Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006)23514-23524). Studies of the human Fc-region-human FcRn complex haveshown that residues 1253, S254, H435, and Y436 are crucial for theinteraction (Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung,Y. A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants ofresidues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 havebeen reported and examined.

In US 2012/0009182 immunoglobulin variants with altered binding toprotein A are reported. The alteration of FcRn binding affinities orserum half-lives of antibodies by mutagenesis is reported in WO2004/035752.

SUMMARY OF THE INVENTION

Herein are reported variant Fc-regions that specifically bind toStaphylococcus protein A and that do or do not bind to human FcRn. Thesevariant Fc-regions contain specific amino acid mutations in theCH2-domain whereas the CH3-domain is not changed with respect to proteinA binding. It has been found that these mutations when used in thehole-chain of a heterodimeric Fc-region allow for the purification ofthe heterodimeric Fc-region, i.e. the separation of the heterodimericFc-region from the homodimeric Fc-region by-product(hole-chain-hole-chain dimer).

One aspect as reported herein is a heterodimeric polypeptide comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction at least a portion of an immunoglobulin hinge region,        which comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain, and a second        polypeptide comprising in N-terminal to C-terminal direction at        least a portion of an immunoglobulin hinge region, which        comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V (hole-chain) and the second polypeptide        comprises the mutations S354C and T366W (knob-chain),    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one, two or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the first polypeptide (hole-chain) comprises themutations

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) T250Q, and/or    -   iv) T256E or T256A.

In one embodiment the first polypeptide (hole-chain) comprises themutations

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) optionally a) T250Q, and/or T256E or T256A, and    -   iv) a) L251A or L251G or L251D, and/or b) H310A or H310G.

In one embodiment the first polypeptide (hole-chain) comprises themutation

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) a) T250Q, and/or T256E or T256A, and.    -   iv) a) L251A or L251G or L251D, and/or b) H310A or H310G.    -   v) optionally a) T307A or T307H or T307Q or T307P, and/or b)        Q311H, and/or c) M252Y, and/or d) S254T.

In one embodiment the second polypeptide (knob-chain) comprises themutation

-   -   i) T250Q, and/or    -   ii) M252Y, and/or    -   iii) S254T, and/or    -   iv) T256E or T256A, and/or    -   v) T307A or T307H or T307Q or T307P, and/or    -   vi) Q311H.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG1 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations L234A and L235A. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations L234A, L235A and P329G.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG4 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations S228P and L235E. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations S228P, L235E and P329G.

In one embodiment the heterodimeric polypeptide is an Fc-region fusionpolypeptide.

In one embodiment the heterodimeric polypeptide is a full-lengthantibody.

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutations I253Aand L314A and a second polypeptide (knob-chain) with the knob-mutations(numbering according to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutations L251Aand L314A and a second polypeptide (knob-chain) with the knob-mutations(numbering according to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutation L251Aand a second polypeptide (knob-chain) with the knob-mutations (numberingaccording to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutation I253Aand a second polypeptide (knob-chain) with the knob-mutations (numberingaccording to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutation L314 Aand a second polypeptide (knob-chain) with the knob-mutations (numberingaccording to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutation H310Aand a second polypeptide (knob-chain) with the knob-mutations (numberingaccording to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutations L251A,I253A and L314A and a second polypeptide (knob-chain) with theknob-mutations (numbering according to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutations L251A,I253A and L314A and a second polypeptide (knob-chain) with in additionto the knob-mutations the mutation M252Y, S254T and T256E (numberingaccording to Kabat).

In one embodiment the full-length antibody comprises a first polypeptide(hole-chain) with in addition to the hole-mutations the mutations I253A,L314A, M428L and N434H and a second polypeptide (knob-chain) with inaddition to the knob-mutations the mutation M252Y, S254T and T256E(numbering according to Kabat).

In one embodiment the full-length antibody further in addition comprisesone or more of the mutations selected from the group comprising S17A,R19A, T57A, T57K, R66A, S70A, Y79A, Q81A, N82aA and S82bA in the heavychain variable domain (numbering according to Kabat). In one embodimentthe full-length antibody comprises one or more of the mutations selectedfrom the group consisting of S17A, R19A, T57A, T57K, R66A, Q81A andN82aA in the heavy chain variable domain and has reduced binding toprotein A compared to an antibody not having these mutations but havingotherwise the identical amino acid sequence (numbering according toKabat). In one embodiment the full-length antibody comprises one or moreof the mutations selected from the group consisting of S70A, Y79A andS82bA in the heavy chain variable domain and has increased binding toprotein A compared to an antibody not having these mutations but havingotherwise the identical amino acid sequence (numbering according toKabat).

In one embodiment the full length antibody is a monospecific antibody.In one embodiment the monospecific antibody is a monovalent monospecificantibody. In one embodiment the monospecific antibody is a bivalentmonospecific antibody.

In one embodiment the full length antibody is a bispecific antibody. Inone embodiment the bispecific antibody is a bivalent bispecificantibody. In one embodiment the bispecific antibody is a tetravalentbispecific antibody.

In one embodiment the full length antibody is a trispecific antibody. Inone embodiment the trispecific antibody is a trivalent trispecificantibody. In one embodiment the trispecific antibody is a tetravalenttrispecific antibody.

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG1, an immunoglobulin hinge region        of the subclass IgG1, an immunoglobulin CH2-domain of the        subclass IgG1 and an immunoglobulin CH3-domain of the subclass        IgG1,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1 and an        immunoglobulin CH3-domain of the subclass IgG1,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, L234A, L235A and P329G and the second        polypeptide comprises the mutations S354C, and T366W, L234A,        L235A and P329G    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG1, an immunoglobulin CH2-domain of the subclass        IgG1 and an immunoglobulin CH3-domain of the subclass IgG1,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1 and an        immunoglobulin CH3-domain of the subclass IgG1,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG1,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, L234A, L235A and P329G and the second        polypeptide comprises the mutations S354C, and T366W, L234A,        L235A and P329G,    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG4, an immunoglobulin hinge region        of the subclass IgG4, an immunoglobulin CH2-domain of the        subclass IgG4 and an immunoglobulin CH3-domain of the subclass        IgG4,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG4, an        immunoglobulin hinge region of the subclass IgG4, an        immunoglobulin CH2-domain of the subclass IgG4 and an        immunoglobulin CH3-domain of the subclass IgG4,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, S228P, L235E and P329G and the second        polypeptide comprises the mutations S354C, and T366W, S228P,        L235E and P329G,    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG4, an immunoglobulin CH2-domain of the subclass        IgG4 and an immunoglobulin CH3-domain of the subclass IgG4,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG4, an        immunoglobulin hinge region of the subclass IgG4, an        immunoglobulin CH2-domain of the subclass IgG4 and an        immunoglobulin CH3-domain of the subclass IgG4,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG4,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, S228P, L235E and P329G and the second        polypeptide comprises the mutations S354C, and T366W, S228P,        L235E and P329G,    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG1, an immunoglobulin hinge region        of the subclass IgG1, an immunoglobulin CH2-domain of the        subclass IgG1, an immunoglobulin CH3-domain of the subclass        IgG1, a peptidic linker and a first scFv,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1, an        immunoglobulin CH3-domain of the subclass IgG1, a peptidic        linker and a second scFv,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen, and the second heavy        chain variable domain and the second light chain variable domain        form a second binding site that specifically binds to a first        antigen, and the first and the second scFv specifically bind to        a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, L234A, L235A and P329G and the second        polypeptide comprises the mutations S354C, and T366W, L234A,        L235A and P329G,    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the heterodimeric polypeptide is a bispecific fulllength antibody comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG1, an immunoglobulin CH2-domain of the subclass        IgG1, an immunoglobulin CH3-domain of the subclass IgG1, a        peptidic linker and a first scFv,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1, an        immunoglobulin CH3-domain of the subclass IgG1, a peptidic        linker and a second scFv,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG1,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen, and the second heavy        chain variable domain and the second light chain variable domain        form a second binding site that specifically binds to a first        antigen, and the first and the second scFv specifically bind to        a second antigen,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A, and Y407V, L234A, L235A and P329G and the second        polypeptide comprises the mutations S354C, and T366W, L234A,        L235A and P329G,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges.

One aspect as reported herein is a method for producing a heterodimericpolypeptide as reported herein comprising the following steps:

-   -   a) cultivating a mammalian cell comprising one or more nucleic        acids encoding the heterodimeric polypeptide,    -   b) recovering the heterodimeric polypeptide from the cultivation        medium, and    -   c) purifying the heterodimeric polypeptide with a protein A        affinity chromatography and thereby producing the dimeric        polypeptide.

One aspect as reported herein is the use of the combination of themutations

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D,

for separating heterodimeric polypeptides from homodimeric polypeptides.

One aspect as reported herein is method of treatment of a patientsuffering from ocular vascular diseases by administering a heterodimericpolypeptide as reported herein to a patient in the need of suchtreatment.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for intravitreal application.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use as a medicament.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for the treatment of vascular eye diseases.

One aspect as reported herein is a pharmaceutical formulation comprisinga heterodimeric polypeptide as reported herein and optionally apharmaceutically acceptable carrier.

For using an antibody that targets/binds to antigens not only present inthe eye but also in the remaining body a short systemic half-live afterpassage of the blood-ocular-barrier from the eye into the blood isbeneficial in order to avoid systemic side effects.

Additionally an antibody that specifically binds to ligands of areceptor is only effective in the treatment of eye-diseases if theantibody-antigen complex is removed from the eye, i.e. the antibodyfunctions as a transport vehicle for receptor ligands out of the eye andthereby inhibits receptor signaling.

One aspect as reported herein is the use of a heterodimeric polypeptideas reported herein for the transport of a soluble receptor ligand fromthe eye over the blood-ocular-barrier into the blood circulation.

One aspect as reported herein is the use of a heterodimeric polypeptideas reported herein for the removal of one or more soluble receptorligands from the eye.

One aspect as reported herein is the use of a heterodimeric polypeptideas reported herein for the treatment of eye diseases, especially ofocular vascular diseases.

One aspect as reported herein is the use of a heterodimeric polypeptideas reported herein for the transport of one or more soluble receptorligands from the intravitreal space to the blood circulation.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use in treating an eye disease.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use in the transport of a soluble receptor ligand from theeye over the blood-ocular-barrier into the blood circulation.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use in the removal of one or more soluble receptor ligandsfrom the eye.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use in treating eye diseases, especially ocular vasculardiseases.

One aspect as reported herein is a heterodimeric polypeptide as reportedherein for use in the transport of one or more soluble receptor ligandsfrom the intravitreal space to the blood circulation.

One aspect as reported herein is a method of treating an individualhaving an ocular vascular disease comprising administering to theindividual an effective amount of a heterodimeric polypeptide asreported herein.

One aspect as reported herein is a method for transporting a solublereceptor ligand from the eye over the blood-ocular-barrier into theblood circulation in an individual comprising administering to theindividual an effective amount of a heterodimeric polypeptide asreported herein to transport a soluble receptor ligand from the eye overthe blood-ocular-barrier into the blood circulation.

One aspect as reported herein is a method the removal of one or moresoluble receptor ligands from the eye in an individual comprisingadministering to the individual an effective amount of a heterodimericpolypeptide as reported herein to remove one or more soluble receptorligands from the eye.

One aspect as reported herein is a method for the transport of one ormore soluble receptor ligands from the intravitreal space to the bloodcirculation in an individual comprising administering to the individualan effective amount of a heterodimeric polypeptide as reported herein totransport of one or more soluble receptor ligands from the intravitrealspace to the blood circulation.

One aspect as reported herein is a method for transporting a solublereceptor ligand from the intravitreal space or the eye over theblood-ocular-barrier into the blood circulation in an individualcomprising administering to the individual an effective amount of aheterodimeric polypeptide as reported herein to transport a solublereceptor ligand from the eye over the blood-ocular-barrier into theblood circulation.

In one embodiment the heterodimeric polypeptide is a bispecificantibody. In one embodiment the bispecific antibody is a bivalentbispecific antibody. In one embodiment the bispecific antibody is atetravalent bispecific antibody.

In one embodiment the heterodimeric polypeptide is a trispecificantibody. In one embodiment the trispecific antibody is a trivalenttrispecific antibody. In one embodiment the trispecific antibody is atetravalent trispecific antibody.

In one embodiment the heterodimeric polypeptide is a CrossMab.

In one embodiment the heterodimeric polypeptide is an Fc-region fusionpolypeptide.

In one embodiment the first polypeptide further comprises the mutationsY349C, T366S, L368A and Y407V and the second polypeptide furthercomprises the mutations S354C and T366W.

In one embodiment the antibody or the Fc-region fusion polypeptide is ofthe subclass IgG1. In one embodiment the antibody or the Fc-regionfusion polypeptide further comprise the mutations L234A and L235A. Inone embodiment the antibody or the Fc-region fusion polypeptide furthercomprise the mutation P329G.

In one embodiment the antibody or the Fc-region fusion polypeptide is ofthe subclass IgG4. In one embodiment the antibody or the Fc-regionfusion polypeptide further comprise the mutations S228P and L235E. Inone embodiment the antibody or the Fc-region fusion polypeptide furthercomprise the mutation P329G.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “about” denotes a range of +/−20% of the thereafter followingnumerical value. In one embodiment the term about denotes a range of+/−10% of the thereafter following numerical value. In one embodimentthe term about denotes a range of +/−5% of the thereafter followingnumerical value.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencealterations. In some embodiments, the number of amino acid alterationsare 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4or less, 3 or less, or 2 or less. In some embodiments, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody, which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “alteration” denotes the mutation (substitution), insertion(addition), or deletion of one or more amino acid residues in a parentantibody or fusion polypeptide, e.g. a fusion polypeptide comprising atleast an FcRn binding portion of an Fc-region, to obtain a modifiedantibody or fusion polypeptide. The term, mutation” denotes that thespecified amino acid residue is substituted for a different amino acidresidue. For example the mutation L234A denotes that the amino acidresidue lysine at position 234 in an antibody Fc-region (polypeptide) issubstituted by the amino acid residue alanine (substitution of lysinewith alanine) (numbering according to the Kabat EU index numberingsystem).

A “naturally occurring amino acid residues” denotes an amino acidresidue from the group consisting of alanine (three letter code: Ala,one letter code: A), arginine (Arg, R), asparagine (Asn, N), asparticacid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid(Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I),leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine(Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T),tryptophane (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

The term “amino acid mutation” denotes the substitution of at least oneexisting amino acid residue with another different amino acid residue(=replacing amino acid residue). The replacing amino acid residue may bea “naturally occurring amino acid residues” and selected from the groupconsisting of alanine (three letter code: Ala, one letter code: A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (asp, D), cysteine(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (glee, G),histidine (his, H), isoleucine (lie, I), leucine (Leu, L), lysine (lees,K), methionine (met, M), phenylalanine (Phe, F), proline (pro, P),serine (seer, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine(try, Y), and valine (Val, V). The replacing amino acid residue may be a“non-naturally occurring amino acid residue”. See e.g. U.S. Pat. No.6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO 2005/35727, WO2005/74524, Chin, J. W., et al., J. Am. Chem. Soc. 124 (2002) 9026-9027;Chin, J. W. and Schultz, P. G., ChemBioChem 11 (2002) 1135-1137; Chin,J. W., et al., PICAS United States of America 99 (2002) 11020-11024;and, Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (all entirelyincorporated by reference herein).

The term “amino acid deletion” denotes the removal of at least one aminoacid residue at a predetermined position in an amino acid sequence.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, multispecific antibodies (e.g. bispecific antibodies,trispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-, and/or protein A and/or FcRn-binding activity.

The term “asymmetric Fc-region” denotes a pair of Fc-region polypeptidesthat have different amino acid residues at corresponding positionsaccording to the Kabat EU index numbering system.

The term “asymmetric Fc-region with respect to FcRn binding” denotes anFc-region that consists of two polypeptide chains that have differentamino acid residues at corresponding positions, whereby the positionsare determined according to the Kabat EU index numbering system, wherebythe different positions affect the binding of the Fc-region to the humanneonatal Fc-receptor (FcRn). For the purpose herein the differencesbetween the two polypeptide chains of the Fc-region in an “asymmetricFc-region with respect to FcRn binding” do not include differences thathave been introduced to facilitate the formation of heterodimericFc-regions, e.g. for the production of bispecific antibodies. Thesedifferences can also be asymmetric, i.e. the two chains have differencesat non-corresponding amino acid residues according to the Kabat EU indexnumbering system. These differences facilitate heterodimerization andreduce homodimerization. Examples of such differences are the so-called“knobs into holes” substitutions (see, e.g., U.S. Pat. No. 7,695,936 andUS 2003/0078385). The following knobs and holes substitutions in theindividual polypeptide chains of an Fc-region of an IgG antibody ofsubclass IgG1 have been found to increase heterodimer formation: 1)Y407T in one chain and T366Y in the other chain; 2) Y407A in one chainand T366W in the other chain; 3) F405A in one chain and T394W in theother chain; 4) F405W in one chain and T394S in the other chain; 5)Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A inone chain and T394W and Y407T in the other chain; 7) T366W and F405W inone chain and T394S and Y407A in the other chain; 8) F405W and Y407A inone chain and T366W and T394S in the other chain; and 9) T366W in onechain and T366S, L368A, and Y407V in the other chain, whereby the lastlisted is especially suited. In addition, changes creating new disulfidebridges between the two Fc-region polypeptide chains facilitateheterodimer formation (see, e.g., US 2003/0078385). The followingsubstitutions resulting in appropriately spaced apart cysteine residuesfor the formation of new intra-chain disulfide bonds in the individualpolypeptide chains of an Fc-region of an IgG antibody of subclass IgG1have been found to increase heterodimer formation: Y349C in one chainand S354C in the other; Y349C in one chain and E356C in the other; Y349Cin one chain and E357C in the other; L351C in one chain and S354C in theother; T394C in one chain and E397C in the other; or D399C in one chainand K392C in the other. Further examples of heterodimerizationfacilitating amino acid changes are the so-called “charge pairsubstitutions” (see, e.g., WO 2009/089004). The following charge pairsubstitutions in the individual polypeptide chains of an Fc-region of anIgG antibody of subclass IgG1 have been found to increase heterodimerformation: 1) K409D or K409E in one chain and D399K or D399R in theother chain; 2) K392D or K392E in one chain and D399K or D399R in theother chain; 3) K439D or K439E in one chain and E356K or E356R in theother chain; 4) K370D or K370E in one chain and E357K or E357R in theother chain; 5) K409D and K360D in one chain plus D399K and E356K in theother chain; 6) K409D and K370D in one chain plus D399K and E357K in theother chain; 7) K409D and K392D in one chain plus D399K, E356K, andE357K in the other chain; 8) K409D and K392D in one chain and D399K inthe other chain; 9) K409D and K392D in one chain and D399K and E356K inthe other chain; 10) K409D and K392D in one chain and D399K and D357K inthe other chain; 11) K409D and K370D in one chain and D399K and D357K inthe other chain; 12) D399K in one chain and K409D and K360D in the otherchain; and 13) K409D and K439D in one chain and D399K and E356K on theother.

The term “binding (to an antigen)” denotes the binding of an antibody toits antigen in an in vitro assay, in one embodiment in a binding assayin which the antibody is bound to a surface and binding of the antigento the antibody is measured by Surface Plasmon Resonance (SPR). Bindingmeans a binding affinity (K_(D)) of 10⁻⁸ M or less, in some embodimentsof 10⁻¹³ to 10⁻⁸ M, in some embodiments of 10⁻¹³ to 10⁻⁹ M.

Binding can be investigated by a BIAcore assay (GE Healthcare BiosensorAB, Uppsala, Sweden). The affinity of the binding is defined by theterms k_(a) (rate constant for the association of the antibody from theantibody/antigen complex), k_(d) (dissociation constant), andK_(D)(k_(d)/k_(a)).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “CH2-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 231 to EUposition 340 (EU numbering system according to Kabat). In one embodimenta CH2 domain has the amino acid sequence of SEQ ID NO: 01: APELLGGPSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ ESTYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.

The term “CH3-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 341 to EUposition 446. In one embodiment the CH3 domain has the amino acidsequence of SEQ ID NO: 02: GQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPG.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “comparable length” denotes that two polypeptides comprise theidentical number of amino acid residues or can be different in length byone or more and up to 10 amino acid residues at most. In one embodimentthe (Fc-region) polypeptides comprise the identical number of amino acidresidues or differ by a number of from 1 to 10 amino acid residues. Inone embodiment the (Fc-region) polypeptides comprise the identicalnumber of amino acid residues or differ by a number of from 1 to 5 aminoacid residues. In one embodiment the (Fc-region) polypeptides comprisethe identical number of amino acid residues or differ by a number offrom 1 to 3 amino acid residues.

“Effector functions” refer to those biological activities attributableto the Fc-region of an antibody, which vary with the antibody class.Examples of antibody effector functions include: C1 q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B-cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc-fusion polypeptide” denotes a fusion of a binding domain(e.g. an antigen binding domain such as a single chain antibody, or apolypeptide such as a ligand of a receptor) with an antibody Fc-regionthat exhibits the desired target-, protein A- and FcRn-binding activity.

The term “Fc-region of human origin” denotes the C-terminal region of animmunoglobulin heavy chain of human origin that contains at least a partof the hinge region, the CH2 domain and the CH3 domain. In oneembodiment, a human IgG heavy chain Fc-region extends from Cys226, orfrom Pro230, to the carboxyl-terminus of the heavy chain. In oneembodiment the Fc-region has the amino acid sequence of SEQ ID NO: 03.However, the C-terminal lysine (Lys447) of the Fc-region may or may notbe present.

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” herein. Specifically the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) is used for the light chainconstant domain CL of kappa and lambda isotype and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3).

The term “FcRn” denotes the human neonatal Fc-receptor. FcRn functionsto salvage IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. The FcRn is a heterodimericprotein consisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of IgG. The interaction between IgG and FcRn isstrictly pH dependent and occurs in a 1:2 stoichiometry, with one IgGbinding to two FcRn molecules via its two heavy chains (Huber, A. H., etal., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in theendosome at acidic pH (pH<6.5) and IgG is released at the neutral cellsurface (pH of about 7.4). The pH-sensitive nature of the interactionfacilitates the FcRn-mediated protection of IgGs pinocytosed into cellsfrom intracellular degradation by binding to the receptor within theacidic environment of endosomes. FcRn then facilitates the recycling ofIgG to the cell surface and subsequent release into the blood streamupon exposure of the FcRn-IgG complex to the neutral pH environmentoutside the cell.

The term “FcRn binding portion of an Fc-region” denotes the part of anantibody heavy chain polypeptide that extends approximately from EUposition 243 to EU position 261 and approximately from EU position 275to EU position 293 and approximately from EU position 302 to EU position319 and approximately from EU position 336 to EU position 348 andapproximately from EU position 367 to EU position 393 and EU position408 and approximately from EU position 424 to EU position 440. In oneembodiment one or more of the following amino acid residues according tothe EU numbering of Kabat are altered F243, P244, P245 P, K246, P247,K248, D249, T250, L251, M252, 1253, S254, R255, T256, P257, E258, V259,T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285,N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305,L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317,E318, Y319, 1336, S337, K338, A339, K340, G341, Q342, P343, R344, E345,P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, 1377, A378,V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391,T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433,N434, H435, Y436, T437, Q438, K439, and S440 (EU numbering).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “full length antibody” denotes an antibody having a structuresubstantially similar to a native antibody structure comprising fourpolypeptides or having heavy chains that contain an Fc-region as definedherein. A full-length antibody may comprise further domains, such ase.g. a scFv or a scFab conjugated to one or more of the chains of thefull-length antibody. These conjugates are also encompassed by the termfull-length antibody.

The term “dimeric polypeptide” denotes a complex comprising at least twopolypeptides that are associated covalently. The complex may comprisefurther polypeptides that are also associated covalently ornon-covalently with the other polypeptides. In one embodiment thedimeric polypeptide comprises two or four polypeptides.

The terms “heterodimer” or “heterodimeric” denote a molecule thatcomprises two polypeptides (e.g. of comparable length), wherein the twopolypeptides have an amino acid sequence that have at least onedifferent amino acid residue in a corresponding position, wherebycorresponding position is determined according to the Kabat EU indexnumbering system.

The terms “homodimer” and “homodimeric” denote a molecule that comprisestwo polypeptides of comparable length, wherein the two polypeptides havean amino acid sequence that is identical in corresponding positions,whereby corresponding positions are determined according to the Kabat EUindex numbering system.

A heterodimeric polypeptide as reported herein is heterodimericdetermined with respect to mutations or properties in focus. Forexample, with respect to FcRn and/or protein A binding (i.e. the focusedon properties) a dimeric polypeptide is homodimeric (i.e. bothpolypeptides of the dimeric polypeptide comprise these mutations) withrespect to the mutations H310A, H433A and Y436A (these mutations are infocus with respect to FcRn and/or protein A binding property of thedimeric polypeptide) but at the same time heterodimeric with respect tothe mutations Y349C, T366S, L368A and Y407V (these mutations are not infocus as these mutations are directed to the heterodimerization of thedimeric polypeptide and not to the FcRn/protein A binding properties) aswell as the mutations S354C and T366W, respectively (the first set iscomprised only in the first polypeptide whereas the second set iscomprised only in the second polypeptide). Further for example, adimeric polypeptide as reported herein can be heterodimeric with respectto the mutations I253A, H310A, H433A, H435A and Y436A (i.e. thesemutations are directed all to the FcRn and/or protein A bindingproperties of the dimeric polypeptide), i.e. one polypeptide comprisesthe mutations I253A, H310A and H435A, whereas the other polypeptidecomprises the mutations H310A, H433A and Y436A.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one that possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework, which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

The term “derived from” denotes that an amino acid sequence is derivedfrom a parent amino acid sequence by introducing alterations at at leastone position. Thus a derived amino acid sequence differs from thecorresponding parent amino acid sequence at at least one correspondingposition (numbering according to Kabat EU index for antibodyFc-regions). In one embodiment an amino acid sequence derived from aparent amino acid sequence differs by one to fifteen amino acid residuesat corresponding positions. In one embodiment an amino acid sequencederived from a parent amino acid sequence differs by one to ten aminoacid residues at corresponding positions. In one embodiment an aminoacid sequence derived from a parent amino acid sequence differs by oneto six amino acid residues at corresponding positions. Likewise aderived amino acid sequence has a high amino acid sequence identity toits parent amino acid sequence. In one embodiment an amino acid sequencederived from a parent amino acid sequence has 80% or more amino acidsequence identity. In one embodiment an amino acid sequence derived froma parent amino acid sequence has 90% or more amino acid sequenceidentity. In one embodiment an amino acid sequence derived from a parentamino acid sequence has 95% or more amino acid sequence identity.

The term “human Fc-region polypeptide” denotes an amino acid sequence,which is identical to a “native” or “wild-type” human Fc-regionpolypeptide. The term “variant (human) Fc-region polypeptide” denotes anamino acid sequence, which is derived from a “native” or “wild-type”human Fc-region polypeptide by virtue of at least one “amino acidalteration”. A “human Fc-region” is consisting of two human Fc-regionpolypeptides. A “variant (human) Fc-region” is consisting of twoFc-region polypeptides, whereby both can be variant (human) Fc-regionpolypeptides or one is a human Fc-region polypeptide and the other is avariant (human) Fc-region polypeptide.

In one embodiment the human Fc-region polypeptide has the amino acidsequence of a human IgG1 Fc-region polypeptide of SEQ ID NO: 03, or of ahuman IgG2 Fc-region polypeptide of SEQ ID NO: 04, or of a human IgG4Fc-region polypeptide of SEQ ID NO: 06 with the mutations as reportedherein. In one embodiment the variant (human) Fc-region polypeptide isderived from an Fc-region polypeptide of SEQ ID NO: 03, or 04, or 06 andhas at least one amino acid mutation compared to the Fc-regionpolypeptide of SEQ ID NO: 03, or 04, or 06. In one embodiment thevariant (human) Fc-region polypeptide comprises/has from about one toabout ten amino acid mutations, and in one embodiment from about one toabout five amino acid mutations. In one embodiment the variant (human)Fc-region polypeptide has at least about 80% homology with a humanFc-region polypeptide of SEQ ID NO: 03, or 04, or 06. In one embodimentthe variant (human) Fc-region polypeptide has least about 90% homologywith a human Fc-region polypeptide of SEQ ID NO: 03, or 04, or 06. Inone embodiment the variant (human) Fc-region polypeptide has at leastabout 95% homology with a human Fc-region polypeptide of SEQ ID NO: 03,or 04, or 06.

The variant (human) Fc-region polypeptide derived from a human Fc-regionpolypeptide of SEQ ID NO: 03, or 04, or 06 is defined by the amino acidalterations that are contained. Thus, for example, the term P329Gdenotes a variant (human) Fc-region polypeptide derived human Fc-regionpolypeptide with the mutation of proline to glycine at amino acidposition 329 relative to the human Fc-region polypeptide of SEQ ID NO:03, or 04, or 06.

A human IgG1 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 03) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with the mutationsL234A, L235A has the following amino acid sequence:

(SEQ ID NO: 07) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A and Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 08) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 09) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 10) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a L234A, L235Aand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 11) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 12) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 13) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P239Gmutation and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 14) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation and S354C, T366W mutation has the following amino acidsequence:

(SEQ ID NO: 15) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G and Y349C, T366S, L368A, Y407V mutations has the following aminoacid sequence:

(SEQ ID NO: 16) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G mutations and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 17) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG4 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 06) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P andL235E mutations has the following amino acid sequence:

(SEQ ID NO: 18) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P, L235Emutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 19) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 20) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQVVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A, Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 21) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 22) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand Y349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 23) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 24) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P239G andY349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 25) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G andS354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 26) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 27) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 28) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., the CDRs)correspond to those of a non-human antibody, and all or substantiallyall of the FRs correspond to those of a human antibody. A humanizedantibody optionally may comprise at least a portion of an antibodyconstant region derived from a human antibody. A “humanized form” of anantibody, e.g., a non-human antibody, refers to an antibody that hasundergone humanization.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and formstructurally defined loops (“hypervariable loops”), and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). HVRs as denoted herein include

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242.);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according to theKabat EU index numbering system (Kabat et al., supra).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one, which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., size exclusionchromatography, ion exchange or reverse phase HPLC). For review ofmethods for assessment of antibody purity, see, e.g., Flatman, S. etal., J. Chrom. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 Da, composed of twoidentical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “peptidic linker” as used herein denotes a peptide with aminoacid sequences, which is in one embodiment of synthetic origin. Thepeptidic linker is in one embodiment a peptide with an amino acidsequence with a length of at least 30 amino acids, in one embodimentwith a length of 32 to 50 amino acids. In one embodiment the peptidiclinker is a peptide with an amino acid sequence with a length of 32 to40 amino acids. In one embodiment the peptidic linker is (G×S)n withG=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8), inone embodiment with x=4, n=6 or 7, in one embodiment with x=4, n=7. Inone embodiment the peptidic linker is (G₄S)₆G₂.

The term “recombinant antibody”, as used herein, denotes all antibodies(chimeric, humanized and human) that are prepared, expressed, created orisolated by recombinant means. This includes antibodies isolated from ahost cell such as a NS0 or CHO cell, or from an animal (e.g. a mouse)that is transgenic for human immunoglobulin genes, or antibodiesexpressed using a recombinant expression vector transfected into a hostcell. Such recombinant antibodies have variable and constant regions ina rearranged form. The recombinant antibodies can be subjected to invivo somatic hypermutation. Thus, the amino acid sequences of the VH andVL regions of the recombinant antibodies are sequences that, whilederived from and related to human germ line VH and VL sequences, may notnaturally exist within the human antibody germ line repertoire in vivo.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies orFc-region fusion polypeptides as reported herein are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in a (antibody)molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding site, four binding sites, and sixbinding sites, respectively, in a (antibody) molecule. The bispecificantibodies as reported herein are in one preferred embodiment“bivalent”.

The term “variable region” or “variable domain” refer to the domain ofan antibody heavy or light chain that is involved in binding of theantibody to its antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of an antibody generally havesimilar structures, with each domain comprising four framework regions(FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T. J. etal. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page91). A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or

VL domain from an antibody that binds the antigen to screen a library ofcomplementary VL or VH domains, respectively (see, e.g., Portolano, S.et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352(1991) 624-628).

The term “ocular vascular disease” includes, but is not limited tointraocular neovascular syndromes such as diabetic retinopathy, diabeticmacular edema, retinopathy of prematurity, neovascular glaucoma, retinalvein occlusions, central retinal vein occlusions, macular degeneration,age-related macular degeneration, retinitis pigmentosa, retinalangiomatous proliferation, macular telangectasia, ischemic retinopathy,iris neovascularization, intraocular neovascularization, comealneovascularization, retinal neovascularization, choroidalneovascularization, and retinal degeneration (see e.g. Garner, A.,Vascular diseases, In: Pathobiology of ocular disease, A dynamicapproach, Garner, A., and Klintworth, G. K., (eds.), 2nd edition, MarcelDekker, New York (1994), pp. 1625-1710).

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The term “with (the) mutation IHH-AAA” as used herein refers to thecombination of the mutations I253A (Ile253A1a), H310A (His310A1a), andH435A (His435A1a) and the term “with (the) mutation HHY-AAA” as usedherein refers to the combination of the mutations H310A (His310A1a),H433A (His433A1a), and Y436A (Tyr436A1a) and the term “with (the)mutation YTE” as used herein refers to the combination of mutationsM252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) in theconstant heavy chain region of IgG1 or IgG4 subclass, wherein thenumbering is according to the Kabat EU index numbering system.

The term “with (the) mutations P329G LALA” as used herein refers to thecombination of the mutations L234A (Leu235A1a), L235A (Leu234A1a) andP329G (Pro329Gly) in the constant heavy chain region of IgG1 subclass,wherein the numbering is according to the Kabat EU index numberingsystem. The term “with (the) mutation SPLE” as used herein refers to thecombination of the mutations S228P (Ser228Pro) and L235E (Leu235Glu) inthe constant heavy chain region of IgG4 subclass, wherein the numberingis according to the Kabat EU index numbering system. The term “with(the) mutation SPLE and P329G” as used herein refers to the combinationof the mutations S228P (Ser228Pro), L235E (Leu235Glu) and P329G(Pro329Gly) in the constant heavy chain region of IgG4 subclass, whereinthe numbering is according to the Kabat EU index numbering system.

II. Compositions and Methods

In one aspect, the invention is based, in part, on the finding thatvariant Fc-regions that specifically bind to Staphylococcus protein Aand that do or do not bind to human FcRn when used in the hole-chain ofa heterodimeric Fc-region allow for the purification of theheterodimeric Fc-region. These variant Fc-regions contain specific aminoacid mutations in the CH2-domain whereas the CH3-domain is not changedwith respect to protein A binding. It has been found that thesemutations when used in the hole-chain of a heterodimeric Fc-region allowfor the purification of the heterodimeric Fc-region, i.e. the separationof the heterodimeric Fc-region from the homodimeric Fc-region by-product(hole-chain-hole-chain dimer).

It has been found that this can be achieved by using the combination ofmutations in the first polypeptide (hole-chain)

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D.

The following table presents an exemplary overview of the amino acidresidues in an Fc-region that are involved in interactions or have beenchanged to modify interactions.

interaction with KiH protein A effect of mutations on residue protein AFcRn knob hole binding FcRn binding Pro238 P238A increase Thr250T250Q/M428L increase Leu251 main-chain contact Met252 hydrophobic M252Wincrease; packing M252Y increase; M252Y/T256Q increase; M252F/T256Dincrease; M252Y/S254T/T256E increase Ile253 main-chain interaction I253Areduction contact; hydrogen bonding; significant binding reduction ifmutated to Ala Ser254 polar S254A reduction; interaction;M252Y/S254T/T256E hydrogen increase bonding Arg255 salt-bridge R255Areduction Thr256 T256A increase; T256Q increase; T256P increase;M252Y/T256Q reduction; M252F/T256D reduction; M252Y/S254T/T256E increasePro257 P257I/Q311I increase; P257I/N434H increase Glu272 E272A increaseAsp280 D280K increase His285 reduction Lys288 K288A reduction;K288A/N434A increase Val305 V305A increase Thr307 T307A increase;T307A/E380A/N434A increase; T307Q/N434A increase; T307Q/N434S increase;T307Q/E380A/N434A increase Val308 V308P/N434A increase Leu309 L309Areduction His310 interaction H310A reduction; H310Q/H433N reductionGln311 polar or Q311A increase; charged P257I/Q311I increase interactionAsp312 D312A increase Leu314 hydrophobic interaction Lys317 K317Aincrease Ala339 A339T increase Tyr349 Y349C Ser354 S354C Thr366 T366WT366S Leu368 L368A Asp376 D376A increase; D376V/N434H increase Ala378A378Q increase Glu380 salt-bridge E380A increase E380A/N434A increase;T307A/E380A/N434A increase; T307Q/E380A/N434A increase Glu382 E382Aincrease Gly385 G385H increase; G385A/Q386P/N389S increase Gln386G385A/Q386P/N389S increase Asn389 G385A/Q386P/N389S increase Tyr407Y407V Ser415 S415A reduction Ser424 S424A increase Met428 M428Lincrease; T250Q/M428L increase Leu432 polar or charged interactionHis433 polar or interaction H433A reduction; charged H310Q/H433Ninteraction; reduction; salt-bridge H433K/N434F/Y436Hincrease;H433R/N434Y/Y436Hincrease; H433K/N434F increase Asn434 hydrogeninteraction N434W/Y/F/A/H bonding; increase; significant K288A/N434Aincrease; binding E380A/N434A increase; reduction if T307A/E380A/N434Areplaced by increase; Ala N434F/Y436H increase;H433K/N434F/Y436Hincrease; H433R/N434Y/Y436Hincrease; H433K/N434Fincrease; P257I/N434H increase; D376V/N434H increase; T307Q/N434Aincrease; T307Q/N434S increase; V308P/N434A increase; T307Q/E380A/N434Aincrease His435 hydrophobic interaction H435R/Y436F H435A reduction;packing; eliminates H435R reduction significant binding to bindingprotein A reduction if mutated to Ala Tyr436 hydrophobic interactionH435R/Y436F Y436A reduction; packing; eliminates N434F/Y436H increase;significant binding to H433K/N434F/Y436Hincrease; binding protein AH433R/N434Y/Y436H reduction if increase replaced by Ala

The modifications as reported herein alter the binding to Staphylococcalprotein A. The mutated residues are all located in the CH2-domain.Although also residues in the CH3-domain do interact with protein A themutations of the residues in the CH2-domain is sufficient to influenceprotein A binding. Thus, in the heterodimeric molecules and antibodiesreported herein both CH3-domain have the same (identical) bindingproperties/characteristics with respect to protein A. Thus, theheterodimeric molecule as reported herein is heterodimeric regardingprotein A binding in the CH2-domain but homodimeric regarding protein Abinding in the CH3-domain.

In one embodiment the combination of mutations as reported herein doesalter or does substantially alter the serum half-life of theheterodimeric polypeptide as compared with a corresponding heterodimericpolypeptide that lacks this combination of mutations.

In one embodiment the combination of mutations further does not alter ordoes not substantially alter the serum half-life of the heterodimericpolypeptide as compared with a corresponding heterodimeric polypeptidethat lacks this combination of mutations.

A. The neonatal Fe-receptor (FeRn)

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvagewild-type IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. It is a heterodimeric proteinconsisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of an antibody of the class IgG. Theinteraction between an antibody of the IgG class and the FcRn is pHdependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibodymolecule can interact with two FcRn molecules via its two heavy chainFc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol. Biol. 230(1993) 1077-1083).

Thus, an IgGs in vitro FcRn binding properties/characteristics areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class different amino acid residues of the heavy chain CH2- andCH3-domain are participating. The amino acid residues interacting withthe FcRn are located approximately between EU position 243 and EUposition 261, approximately between EU position 275 and EU position 293,approximately between EU position 302 and EU position 319, approximatelybetween EU position 336 and EU position 348, approximately between EUposition 367 and EU position 393, at EU position 408, and approximatelybetween EU position 424 and EU position 440. More specifically thefollowing amino acid residues according to the EU numbering of Kabat areinvolved in the interaction between the Fc-region and the FcRn: F243,P244, P245 P, K246, P247, K248, D249, T250, L251, M252, 1253, S254,R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279,D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292,E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312,W313, L314, N315, G316, K317, E318, Y319, 1336, S337, K338, A339, K340,G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373,P374, S375, D376, 1377, A378, V379, E380, W381, E382, S383, N384, G385,Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428,H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439, andS440.

Site-directed mutagenesis studies have proven that the critical bindingsites in the Fc-region of IgGs for FcRn are Histidine 310, Histidine435, and Isoleucine 253 and to a lesser extent Histidine 433 andTyrosine 436 (see e.g. Kim, J. K., et al., Eur. J. Immunol. 29 (1999)2819-2825; Raghavan, M., et al., Biochem. 34 (1995) 14649-14657;Medesan, C., et al., J Immunol. 158 (1997) 2211-2217).

Methods to increase IgG binding to FcRn have been performed by mutatingIgG at various amino acid residues: Threonine 250, Methionine 252,Serine 254, Threonine 256, Threonine 307, Glutamic acid 380, Methionine428, Histidine 433, and Asparagine 434 (see Kuo, T. T., et al., J. Clin.Immunol. 30 (2010) 777-789).

In some cases antibodies with reduced half-life in the blood circulationare desired. For example, drugs for intravitreal application should havea long half-live in the eye and a short half-life in the bloodcirculation of the patient. Such antibodies also have the advantage ofincreased exposure to a disease site, e.g. in the eye.

Different mutations that influence the FcRn binding and therewith thehalf-live in the blood circulation are known. Fc-region residuescritical to the mouse Fc-region—mouse FcRn interaction have beenidentified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., etal. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433, N434,and H435 (EU numbering according to Kabat) are involved in theinteraction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533-2536;Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Kim, J. K., et al.,Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310, and H435 werefound to be critical for the interaction of human Fc with murine FcRn(Kim, J. K., et al., Eur. J. Immunol. 29 (1999) 2819-2855). ResiduesM252Y, S254T, T256E have been described by Dall'Acqua et al. to improveFcRn binding by protein-protein interaction studies (Dall'Acqua, W. F.,et al. J. Biol. Chem. 281 (2006) 23514-23524). Studies of the humanFc-human FcRn complex have shown that residues 1253, S254, H435, andY436 are crucial for the interaction (Firan, M., et al., Int. Immunol.13 (2001) 993-1002; Shields, R. L., et al., J. Biol. Chem. 276 (2001)6591-6604). In Yeung, Y. A., et al. (J. Immunol. 182 (2009) 7667-7671)various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and424 to 437 have been reported and examined. Exemplary mutations andtheir effect on FcRn binding are listed in the following Table.

TABLE effect on half-live in the mutation FcRn binding circulationreference H285 reduced reduced Kim, J. K., H310Q/H433N (murine) (inmouse) Scand. J. (murine IgG1) Immunol. 40 (1994) 457- 465 I253A reducedreduced Ghetie, V. and H310A (murine) (in mouse) Ward, E. S., H435AImmunol. H436A Today 18 (murine IgG1) (1997) 592- 598 T252L/T254S/T256Fincreased increased Ghetie, V. and T252A/T254S/T256A (murine) (in mouse)Ward, E. S., (murine IgG1) Immunol. Today 18 (1997) 592- 598 I253Areduced reduced Medesan, C., H310A (murine) (in mouse) et al., J. H435AImmunol. 158 H436A (1997) 2211- H433A/N434Q 2217 (murine IgG1) I253Areduced reduced Kim, J. K., H310A H310A: <0.1 (in mouse) Eur. J. H435Arel. binding to Immunol. 29 H435R muFcRn (1999) 2819- (human IgG1)(murine) 2825 H433A 1.1 rel. binding Kim, J. K., (human IgG1) to muFcRn,Eur. J. 0.4 rel. binding Immunol. 29 Hu FcRn (1999) 2819- (murine) 2825I253A reduced <0.1 reduced Shields, R. L., S254A relative et al., J.Biol. H435A binding to Chem. 276 Y436A huFcRn (2001) 6591- (human IgG1)6604 R255A reduced reduced Shields, R. L., K288A (human) et al., J.Biol. L309A Chem. 276 S415A (2001) 6591- H433A 6604 (human IgG1) P238Aincreased increased Shields, R. L., T256A (human) et al., J. Biol. E272AChem. 276 V305A (2001) 6591- T307A 6604 Q311A D312A K317A D376A A378QE380A E382A S424A N434A K288A/N434A E380A/N434A T307A/E380A/N434A (humanIgG1) H435A reduced <0.1 reduced Firan, M., et (humanized IgG1) rel.al., Int. binding to Immunol. 13 huFcRn (2001) 993- 1002 I253A (nobinding) increased reduced Dall'Acqua, J. M252W (murine and (in mouse)Immunol. 169 M252Y human) (2002) 5171- M252Y/T256Q 5180 M252F/T256DN434F/Y436H M252Y/S254T/T256E G385A/Q386P/N389S H433K/N434F/Y436HH433R/N434Y/Y436H G385R/Q386T/P387R/N389P M252Y/S254T/T256E/H433K/N434F/Y436H M252Y/S254T/T256E/G385R/ Q386T/P387R/N389P (human IgG1)M428L increased increased Hinton, P. R., T250Q/M428L (human) (in monkey)et al., J. Biol. (human IgG2) Chem. 279 (2004) 6213- 6216M252Y/S254T/T256E + increased increased Vaccaro, C., et H433K/N434F(human) (in mouse) al., Nat. (human IgG) Biotechnol. 23 (2005) 1283-1288 T307A/E380A/N434A increased increased in Pop, L. M., et (chimericIgG1) transgenic mouse al., Int. Immunopharmacol. 5 (2005) 1279-1290T250Q increased increased in Petkova, S. B., E380A (human) transgenicmouse et al., Int. M428L Immunol 18 N434A (2006) 1759- K288A/N434A 1769E380A/N434A T307A/E380A/N434A (human IgG1) I253A reduced reduced inPetkova, S. B., (human IgG1) (human) transgenic mouse et al., Int.Immunol 18 (2006) 1759- 1769 S239D/A330L/I332E increased increased inDall'Acqua, M252Y/S254T/T256E (human and Cynomolgus W. F., et al., J.(humanized) Cynomolgus) Biol. Chem. 281 (2006) 23514-23524 T250Qincreased increased in Rhesus Hinton, P. R., M428L (human) apes et al.,J. T250Q/M428L Immunol. 176 (human IgG1) (2006) 346- 356 T250Q/M428Lincreased no change in Datta- P257I/Q311I (mouse and Cynomolgus Mannan,A., et (humanized IgG1) Cynomolgus) increased in mouse al., J. Biol.Chem. 282 (2007) 1709- 1717 P257I/Q311I increased reduced in mice Datta-P257I/N434H at pH 6 P257I/N434H Mannan, A., et D376V/N434H (human,reduced in al., Drug (humanized IgG1) Cynomolgus, Cynomolgus Metab.mouse) Dispos. 35 (2007) 86-94 abrogate FcRn binding: increased andreducing the Ropeenian, I253 reduced binding ability of D. C. and H310IgG for FcRn Akilesh, S., H433 reduces its serum Nat. Rev. H435persistence; a Immunol. 7 reduce FcRn binding: higher-affinity (2007)715- Y436 FcRn-IgG 725 increased FcRn binding: interaction prolongs T250the half-lives of IgG N252 and Fc-coupled S254 drugs in the serum T256T307 M428 N434 N434A increased increased in Yeung, Y. A., T307Q/N434A(Cynomolgus Cynomolgus et al., Cancer T307Q/N434S monkey) monkey Res. 70(2010) V308P/N434A 3269-3277 T307Q/E380A/N434A (human IgG1) 256Pincreased at WO 2011/ 280K neutral pH 122011 339T 385H 428L 434W/Y/F/A/H(human IgG)

The results of a symmetric engineering of an IgG1 Fc-region to influenceFcRn binding is shown in the following table (alignment of mutations andretention time on an FcRn-affinity chromatography column).

TABLE FcRn- affinity FcRn- FcRn- FcRn- column effector function bindingbinding binding retention influencing influencing influencinginfluencing time mutations mutation 1 mutation 2 mutation 3 [min]L234A/L235A/P329G — — — 45.3 L234A/L235A/P329G I253A H310A H435A 2.3L234A/L235A/P329G I253A — — 2.7 L234A/L235A/P329G — H310A — 2.4L234A/L235A/P329G — — H435A 2.7 L234A/L235A/P329G I253A H310A — 2.3L234A/L235A/P329G I253A — H435A 2.3 L234A/L235A/P329G — H310A H435A 2.4L234A/L235A/P329G — H310A Y436A 2.3 L234A/L235A/P329G H310A H433A Y436A2.4 L234A/L235A/P329G — — Y436A 41.3

Retention times below 3 minutes correspond to no binding as thesubstance is in the flow-through (void peak).

The single mutation H310A is the most silent symmetrical mutation todelete any FcRn-binding.

The symmetric single mutation I253A and H435A result in a relative shiftof retention time of 0.3 to 0.4 min. This can be generally regarded as anon-detectable binding.

The single mutation Y436A results in detectable interaction strength tothe FcRn affinity column. Without being bound by this theory thismutation could have an effect on FcRn mediated in vivo half-life, whichcan be differentiated from a zero interaction such as the combination ofthe I253A, H310A and H435A mutations (IHH-AAA mutation).

The results obtained with a symmetrically modified anti-HER2 antibodyare presented in the following table (see WO 2006/031370 for reference).

TABLE retention time mutation [min] I253H no binding M252D no bindingS254D no binding R255D 41.4 M252H 43.6 K288E 45.2 L309H 45.5 E258H 45.6T256H 46.0 K290H 46.2 D98E 46.2 wild-type 46.3 K317H 46.3 Q311H 46.3E430H 46.4 T307H 47.0 N434H 52.0

B. Ocular Vascular Diseases

Ocular vascular diseases are any pathological condition characterized byaltered or unregulated proliferation and invasion of new blood vesselsinto the structures of ocular tissues such as the retina or cornea.

In one embodiment the ocular vascular disease is selected from the groupconsisting of wet age-related macular degeneration (wet AMD), dryage-related macular degeneration (dry AMD), diabetic macular edema(DME), cystoid macular edema (CME), non-proliferative diabeticretinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoidmacular edema, vasculitis (e.g. central retinal vein occlusion),papilloedema, retinitis, conjunctivitis, uveitis, choroiditis,multifocal choroiditis, ocular histoplasmosis, blepharitis, dry eye(Sjogren's disease) and other ophthalmic diseases wherein the eyedisease or disorder is associated with ocular neovascularization,vascular leakage, and/or retinal edema.

The antibody comprising the heterodimeric polypeptide as reported hereinis useful in the prevention and treatment of wet AMD, dry AMD, CME, DME,NPDR, PDR, blepharitis, dry eye and uveitis, in one preferred embodimentwet AMD, dry AMD, blepharitis, and dry eye, also in one preferredembodiment CME, DME, NPDR and PDR, also in one preferred embodimentblepharitis, and dry eye, in particular wet AMD and dry AMD, and alsoparticularly wet AMD.

In some embodiments, the ocular vascular disease is selected from thegroup consisting of wet age-related macular degeneration (wet AMD),macular edema, retinal vein occlusions, retinopathy of prematurity, anddiabetic retinopathy.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, Sjogren's disease, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegener's sarcoidosis, Scleritis, Steven's Johnson disease,periphigoid radial keratotomy, and corneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, retinitispigmentosa, retina edema (including macular edema), Eale's disease,Bechet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Best's disease, myopia, optic pits,Stargart's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications.

Other diseases include, but are not limited to, diseases associated withrubeosis (neovascularization of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Retinopathy of prematurity (ROP) is a disease of the eye that affectsprematurely born babies. It is thought to be caused by disorganizedgrowth of retinal blood vessels, which may result in scarring andretinal detachment. ROP can be mild and may resolve spontaneously, butmay lead to blindness in serious cases. As such, all preterm babies areat risk for ROP, and very low birth weight is an additional risk factor.Both oxygen toxicity and relative hypoxia can contribute to thedevelopment of ROP.

Macular degeneration is a medical condition predominantly found inelderly adults in which the center of the inner lining of the eye, knownas the macula area of the retina, suffers thinning, atrophy, and in somecases, bleeding. This can result in loss of central vision, whichentails inability to see fine details, to read, or to recognize faces.According to the American Academy of Ophthalmology, it is the leadingcause of central vision loss (blindness) in the United States today forthose over the age of fifty years. Although some macular dystrophiesthat affect younger individuals are sometimes referred to as maculardegeneration, the term generally refers to age-related maculardegeneration (AMD or ARMD).

Age-related macular degeneration begins with characteristic yellowdeposits in the macula (central area of the retina which providesdetailed central vision, called fovea) called drusen between the retinalpigment epithelium and the underlying choroid. Most people with theseearly changes (referred to as age-related maculopathy) have good vision.People with drusen can go on to develop advanced AMD. The risk isconsiderably higher when the drusen are large and numerous andassociated with disturbance in the pigmented cell layer under themacula. Large and soft drusen are related to elevated cholesteroldeposits and may respond to cholesterol lowering agents or the RheoProcedure.

Advanced AMD, which is responsible for profound vision loss, has twoforms: dry and wet. Central geographic atrophy, the dry form of advancedAMD, results from atrophy to the retinal pigment epithelial layer belowthe retina, which causes vision loss through loss of photoreceptors(rods and cones) in the central part of the eye. While no treatment isavailable for this condition, vitamin supplements with high doses ofantioxidants, lutein and zeaxanthin, have been demonstrated by theNational Eye Institute and others to slow the progression of dry maculardegeneration and in some patients, improve visual acuity.

Retinitis pigmentosa (RP) is a group of genetic eye conditions. In theprogression of symptoms for RP, night blindness generally precedestunnel vision by years or even decades. Many people with RP do notbecome legally blind until their 40s or 50s and retain some sight alltheir life. Others go completely blind from RP, in some cases as earlyas childhood. Progression of RP is different in each case. RP is a typeof hereditary retinal dystrophy, a group of inherited disorders in whichabnormalities of the photoreceptors (rods and cones) or the retinalpigment epithelium (RPE) of the retina lead to progressive visual loss.Affected individuals first experience defective dark adaptation ornyctalopia (night blindness), followed by reduction of the peripheralvisual field (known as tunnel vision) and, sometimes, loss of centralvision late in the course of the disease.

Macular edema occurs when fluid and protein deposits collect on or underthe macula of the eye, a yellow central area of the retina, causing itto thicken and swell. The swelling may distort a person's centralvision, as the macula is near the center of the retina at the back ofthe eyeball. This area holds tightly packed cones that provide sharp,clear central vision to enable a person to see form, color, and detailthat is directly in the line of sight. Cystoid macular edema is a typeof macular edema that includes cyst formation.

C. The current invention

It has been found that one mutation one-sided in one Fc-regionpolypeptide is sufficient to influence the binding significantly. Themore mutations are introduced into the Fc-region the more the binding toStaphylococcal protein A and/or the FcRn is changed, i.e. weakened orstrengthened.

Herein is reported a method to deplete the hole-hole mispairingby-product occurring in the production of knob-into-hole (KiH)bispecific antibodies by CH2 domain design.

The amino acid positions L251, 1253, H310, L314 in the CH2 domain areinteracting with protein A and the human neonatal Fc receptor (FcRn).

By introducing an amino acid exchange at one or more of these positionsin the so call hole-chain of a KiH bispecific antibody to an A, G or Dthe binding property to protein A and FcRn can be silenced.

In one preferred embodiment the CH2 domain comprises the mutations i)I253A or I253G, and ii) L314A or L314G or L314D.

By this design the hole-hole mispairing by-product can no longer bindprotein A and FcRn (no interaction possible with both heavy chains).Thereby the hole-hole mispairing by-product will not bind to a protein Aand/or FcRn affinity chromatography column. Thus, the hole-holemispairing by-product will at least elute earlier, i.e. in a separateddetached peak, or will be not bind at all and can be found in theflow-through of the protein A or FcRn affinity column.

To compensate the impaired FcRn binding properties the surrounding aminoacids can be improved by introducing to the hole side a T250Q and/or aT256E and/or a T307H mutation, either alone or in a single, double ortriple combination.

hole chain amino acid knob chain amino acid amino acid and positionmutation mutation resulting in a decrease of FcRn binding in the finalbispecific molecule L251 A, G, D L I253 A, G I H310 A, G H L314 A, G, DL resulting in a decrease in protein A binding with compensation of FcRnbinding on the hole side in the final bispecific molecule; can be usedsingle or in combination T250 Q T M252 Y M S254 T S T256 E, A T T307 A,H, Q, P T Q311 H Q resulting in a decrease in protein A binding withcompensation of FcRn binding on the knob side in the final bispecificmolecule; can be used single or in combination T250 T Q M252 M Y S254 ST T256 T E, A T307 T A, H, Q, P Q311 Q H

An overview on FcRn binding engineering is provided in the followingTable:

FcRn binding FcRn binding improved by decreased by protein A protein GPosition mutation to mutation to binding binding CH2 T250 Q L251 D yesyes M252 Y L, D, (H) yes yes I253 A yes yes S254 T A, D yes yes R255 A,D T256 E, A E258 (H) K288 E T307 A, H, Q, P, H L309 A, H H310 A Q311 HL314 D yes no CH3 M428 L (E) yes E430 H L432 D yes yes H433 A no yesN434 A, W, Y, H yes yes H435 A yes Y436 A no yes

One aspect as reported herein is a heterodimeric polypeptide comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction at least a portion of an immunoglobulin hinge region,        which comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain, and a second        polypeptide comprising in N-terminal to C-terminal direction at        least a portion of an immunoglobulin hinge region, which        comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain,    -   wherein the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V (hole-chain) and the second polypeptide        comprises the mutations S354C and T366W (knob-chain),    -   and    -   wherein the first polypeptide (hole-chain) comprises the        mutations        -   i) I253A or I253G, and        -   ii) L314A or L314G or L314D,    -   and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges,    -   and    -   wherein the CH3-domain of the first polypeptide and the        CH3-domain of the second polypeptide both bind or both do not        bind to protein A    -   (numbering according to the Kabat EU index).

In one embodiment the first polypeptide (hole-chain) comprises themutations

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) T250Q, and/or    -   iv) T256E or T256A.

In one embodiment the first polypeptide (hole-chain) comprises themutations

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) optionally a) T250Q, and/or T256E or T256A, and.    -   iv) a) L251A or L251G or L251D, and/or b) H310A or H310G.

In one embodiment the first polypeptide (hole-chain) comprises themutation

-   -   i) I253A or I253G, and    -   ii) L314A or L314G or L314D, and    -   iii) a) T250Q, and/or T256E or T256A, and.    -   iv) a) L251A or L251G or L251D, and/or b) H310A or H310G.    -   v) optionally a) T307A or T307H or T307Q or T307P, and/or b)    -   Q311H, and/or c) M252Y, and/or d) S254T.

In one embodiment the second polypeptide (knob-chain) comprises themutation

-   -   i) T250Q, and/or    -   ii) M252Y, and/or    -   iii) S254T, and/or    -   iv) T256E or T256A, and/or    -   v) T307A or T307H or T307Q or T307P, and/or    -   vi) Q311H.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG1 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations L234A and L235A. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations L234A, L235A and P329G.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG4 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations S228P and L235E. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations S228P, L235E and P329G.

In one embodiment the heterodimeric polypeptide is an Fc-region fusionpolypeptide.

In one embodiment the heterodimeric polypeptide is a full-lengthantibody. In one embodiment the

In one embodiment the full length antibody is a monospecific antibody.In one embodiment the monospecific antibody is a monovalent monospecificantibody. In one embodiment the monospecific antibody is a bivalentmonospecific antibody.

In one embodiment the full length antibody is a bispecific antibody. Inone embodiment the bispecific antibody is a bivalent bispecificantibody. In one embodiment the bispecific antibody is a tetravalentbispecific antibody.

In one embodiment the full length antibody is a trispecific antibody. Inone embodiment the trispecific antibody is a trivalent trispecificantibody. In one embodiment the trispecific antibody is a tetravalenttrispecific antibody.

One aspect as reported herein is an antibody comprising theheterodimeric polypeptide (variant human IgG class Fc-region) asreported herein.

The Fc-region (heterodimeric polypeptide) as reported herein whencontained in a full length antibody confers the above describedcharacteristics to the molecule.

Antibodies, e.g. full-length antibodies or CrossMabs, can comprise avariant (human) human IgG class Fc-region as reported herein.

The heterodimeric polypeptides have due to the mutations the propertiesof not binding to Staphylococcal protein A in one chain (the hole-chain)and of binding to Staphylococcal protein A in the other chain (theknob-chain).

Thus, these antibodies can be purified, i.e. separated from unwantedhole-chain dimeric by-products by using conventional protein A affinitymaterials, such as MabSelectSure. It is not required to use highlysophisticated but species limited affinity materials, such as e.g.KappaSelect, which is only useable with antibodies comprising a lightchain of the kappa subclass. Additionally it is not required to adoptthe purification method if a modification/exchange of the light chainsubclass is made.

One aspect as reported herein is a method for producing a heterodimericpolypeptide as reported herein comprising the following steps:

-   -   a) cultivating a mammalian cell comprising one or more nucleic        acids encoding a heterodimeric polypeptide as reported herein,    -   b) recovering the heterodimeric polypeptide from the cultivation        medium, and    -   c) purifying the heterodimeric polypeptide with a protein A        affinity chromatography and thereby producing the dimeric        polypeptide.

One aspect as reported herein is the use of the combination of mutationsi) I253A or I253G, and ii) L314A or L314G or L314D for separatingheterodimeric polypeptides from homodimeric polypeptides.

One aspect as reported herein is a bispecific antibody providing ease ofisolation/purification comprising immunoglobulin heavy chain Fc-regionsthat are differentially modified, wherein at least one of themodifications results in i) a differential affinity of the bispecificantibody for protein A, and the heterodimeric bispecific antibody isisolable from a disrupted cell, from medium, or from a mixture ofantibodies based on its affinity for protein A.

In one embodiment the bispecific antibody elutes at a pH value above pH4.0.

In one embodiment the bispecific antibody is isolated using a protein Aaffinity chromatography and a pH gradient or pH step, wherein the pHgradient or pH step includes the addition of a salt. In a specificembodiment, the salt is present at a concentration of about 0.5 molar toabout 1 molar. In one embodiment, the salt is selected from the groupconsisting of lithium, sodium, and potassium salts of acetate; sodiumand potassium bicarbonates; lithium, sodium, and potassium carbonates;lithium, sodium, potassium, and magnesium chlorides; sodium andpotassium fluorides; sodium, potassium, and calcium nitrates; sodium andpotassium phosphates; and calcium and magnesium sulfates. In oneembodiment the salt is a halide salt of an alkaline metal or alkalineearth metal. In one preferred embodiment the salt is sodium chloride.

In one aspect the dimeric polypeptide comprises a first polypeptide thatis modified as reported herein and a second polypeptide that is notmodified regarding protein A or FcRn binding, so as to form aheterodimeric polypeptide, wherein the differential modification resultsin the dimeric polypeptide eluting from a protein A affinity material at0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, or 1.4 pH unit(s) higher than acorresponding dimeric polypeptide that lacks the differentialmodification. In one embodiment, the differentially modified dimericpolypeptide elutes at a pH of 4 or higher, whereas the unmodifieddimeric polypeptide elutes at a pH of 3.5 or lower. In one embodiment,the differentially modified dimeric polypeptide elutes at a pH of about4, whereas the unmodified dimeric polypeptide elutes at a pH of about2.8-3.5, 2.8-3.2, or 2.8-3. In these embodiments, “unmodified” refers tolack of the modification i) I253A and I253G, and ii) L314A and L314G andL314D (Kabat EU index numbering system) in both of the polypeptides.

For chromatographic runs the addition of 0.5 molar to 1 molar salt (e.g.NaCl) may improve the separation of homodimeric polypeptide andheterodimeric polypeptide, especially if derived from the human IgG1subclass. The addition of salt to the elution solution increasing the pHvalue can broaden the pH range for elution such that e.g. a pH stepgradient could successfully separate the two species.

Accordingly, in one embodiment a method for separating a bispecificantibody comprising a heterodimeric IgG Fc-region with one chaincomprising mutations as reported herein, comprises a step of employing apH gradient in the presence of a salt. In one embodiment, the salt ispresent at a concentration sufficient to maximize the pH differencebetween elution from a protein A chromatography material of an IgGFc-region homodimer and an IgG Fc-region heterodimer. In one embodimentthe salt is present at a concentration of about 0.5 molar to about 1molar. In one embodiment the salt is a salt of an alkaline metal or analkaline earth metal and a halogen. In one embodiment the salt is achloride salt of an alkaline metal or an alkaline earth metal, such ase.g. NaCl, KCl, LiCl, CaCl₂, or MgCl₂. In one embodiment the pH gradientis from about pH 4 to about pH 5. In one embodiment the gradient is alinear gradient. In one embodiment, the pH gradient is a step gradient.In one embodiment the method comprises applying to an equilibratedprotein A affinity column a solution of about pH 4. In one embodimentthe bispecific antibody comprising the heterodimeric IgG Fc-region withrespect to the modifications as reported herein elutes from the proteinA affinity chromatography material in one or more fractionssubstantially free of non-heterodimeric bispecific antibody.

The heterodimeric polypeptide as reported herein is produced byrecombinant means. Thus, one aspect of the current invention is anucleic acid encoding the heterodimeric polypeptide as reported hereinand a further aspect is a cell comprising the nucleic acid encoding theheterodimeric polypeptide as reported herein.

Methods for recombinant production are widely known in the state of theart and comprise protein expression in prokaryotic and eukaryotic cellswith subsequent isolation of the heterodimeric polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the heterodimeric polypeptides as aforementioned in a host cell,nucleic acids encoding the respective first and second polypeptides areinserted into expression vectors by standard methods. Expression isperformed in appropriate prokaryotic or eukaryotic host cells like CHOcells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells,yeast, or E. coli cells, and the heterodimeric polypeptide is recoveredfrom the cells (cultivation supernatant or cells after lysis).

General methods for recombinant production of antibodies are well knownin the state of the art and described, for example, in the reviewarticles of Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202;Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R. G., Drug Res. 48(1998) 870-880.

Accordingly one aspect as reported herein is a method for the productionof a heterodimeric polypeptide as reported herein, comprising the stepsof

-   -   a) transforming a host cell with one or more vectors comprising        nucleic acid molecules encoding the heterodimeric polypeptide as        reported herein,    -   b) culturing the host cell to express the heterodimeric        polypeptide, and c) recovering the heterodimeric polypeptide        from the culture and thereby producing the heterodimeric        polypeptide.

In one embodiment the recovering step under c) includes the use of animmunoglobulin Fc-region specific capture reagent. In one embodimentthis Fc-region specific capture reagent is used in abind-and-elute-mode. Examples of such

Fc-region specific capture reagents are e.g. Staphylococcus proteinA-based affinity chromatography columns, which are based on a highlyrigid agarose base matrix that allows high flow rates and low backpressure at large scale. They feature a ligand that binds to theheterodimeric polypeptide, i.e. its Fc-region. The ligands are attachedto the matrix via a long hydrophilic spacer arm to make it easilyavailable for binding to the target molecule.

The heterodimeric polypeptides as reported herein are suitably separatedfrom the culture medium by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography.

DNA and RNA encoding the monoclonal antibodies are readily isolated andsequenced using conventional procedures. Once isolated, the DNA may beinserted into expression vectors, which are then transfected into hostcells such as HEK 293 cells, CHO cells, or myeloma cells that do nototherwise produce heterodimeric polypeptides, to obtain the synthesis ofrecombinant monoclonal heterodimeric polypeptides in the host cells.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art (see Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987)). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-Sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

One aspect of the invention is a pharmaceutical formulation comprising aheterodimeric polypeptide as reported herein. Another aspect of theinvention is the use of a heterodimeric polypeptide as reported hereinfor the manufacture of a pharmaceutical formulation. A further aspect ofthe invention is a method for the manufacture of a pharmaceuticalformulation comprising a heterodimeric polypeptide as reported herein.In another aspect, the present invention provides a formulation, e.g. apharmaceutical formulation, containing a heterodimeric polypeptide asreported herein, formulated together with a pharmaceutical carrier.

A formulation as reported herein can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. To administer a compound of the invention by certainroutes of administration, it may be necessary to coat the compound with,or co-administer the compound with, a material to prevent itsinactivation. For example, the compound may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

Many possible modes of delivery can be used, including, but not limitedto intraocular application or topical application. In one embodiment theapplication is intraocular and includes, but it's not limited tosubconjunctival injection, intracanieral injection, injection into theanterior chamber via the termporai limbus, intrastromal injection,intracorneal injection, subretinal injection, aqueous humor injection,subtenon injection or sustained delivery device, intravitreal injection(e.g., front, mid or back vitreal injection). In one embodiment theapplication is topical and includes, but it's not limited to eye dropsto the cornea.

In one embodiment the heterodimeric polypeptide as reported herein orthe pharmaceutical formulation as reported herein is administered viaintravitreal application, e.g. via intravitreal injection. This can beperformed in accordance with standard procedures known in the art. See,e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-3276;Russelakis-Cameiro et al., Neuropathol. Appl. Neurobiol. 25 (1999)196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185.

In some embodiments, therapeutic kits of the invention can contain oneor more doses of a heterodimeric polypeptide as reported herein presentin a pharmaceutical formulation as described herein, a suitable devicefor intravitreal injection of the pharmaceutical formulation, and aninstruction detailing suitable subjects and protocols for carrying outthe injection. In these embodiments, the formulations are typicallyadministered to the subject in need of treatment via intravitrealinjection. This can be performed in accordance with standard proceduresknown in the art (see, e.g., Ritter et al., J. Clin. Invest. 116 (2006)3266-3276; Russelakis-Cameiro et al., Neuropathol. Appl. Neurobiol. 25(1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185).

The formulation may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the formulations. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents, which delay absorption,such as aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds asreported herein, which may be used in a suitable hydrated form, and/orthe pharmaceutical formulations as reported herein, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalformulation as reported herein may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The formulation must be sterile and fluid to the extent that theformulation is deliverable by syringe. In addition to water, the carrierin one preferred embodiment is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

The formulation can comprise an ophthalmic depot formulation comprisingan active agent for subconjunctival administration. The ophthalmic depotformulation comprises microparticles of essentially pure active agent,e.g., a heterodimeric polypeptide as reported herein. The microparticlescomprising a heterodimeric polypeptide as reported herein can beembedded in a biocompatible pharmaceutically acceptable polymer or alipid-encapsulating agent. The depot formulations may be adapted torelease all of substantially all the active material over an extendedperiod of time. The polymer or lipid matrix, if present, may be adaptedto degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent. Upon injection, the polymer forms a depot at the injections site,e.g. by gelifying or precipitating.

Another aspect of the invention is a heterodimeric polypeptide asreported herein for use in the treatment of ocular vascular diseases.

One embodiment of the invention is a heterodimeric polypeptide asreported herein for use in the treatment of ocular vascular diseases.

Another aspect of the invention is the pharmaceutical formulation foruse in the treatment of ocular vascular diseases.

Another aspect of the invention is the use of a heterodimericpolypeptide as reported herein for the manufacture of a medicament forthe treatment of ocular vascular disease.

Another aspect of the invention is method of treatment of patientsuffering from ocular vascular diseases by administering a heterodimericpolypeptide as reported herein to a patient in the need of suchtreatment.

It is herewith expressly stated that the term “comprising” as usedherein comprises the term “consisting of”. Thus, all aspects andembodiments that contain the term “comprising” are likewise disclosedwith the term “consisting of”.

D. Modifications

In a further aspect, a heterodimeric polypeptide according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-6 below:

1. Antibody Affinity

In one embodiment, Kd is measured using a BIACORE® surface plasmonresonance assay. For example, an assay using a BIACORE®-2000 or aBIACORE®-3000 (GE Healthcare Inc., Piscataway, N.J.) is performed at 25°C. with immobilized binding partner CM5 chips at ˜10 response units(RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5,GE Healthcare Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Binding partner is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/mL(˜0.2 μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled binding partner.Following the injection of the binding partner, 1 M ethanolamine isinjected to block non-reacted groups. For kinetics measurements,two-fold serial dilutions of the dimeric polypeptide containing fusionpolypeptide or antibody (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μL/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on) (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Chimeric and Humanized Antibodies

In certain embodiments, a heterodimeric polypeptide as reported hereinis comprised in a chimeric antibody. Certain chimeric antibodies aredescribed, e.g., in U.S. Pat. No. 4,816,567; and Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In a furtherexample, a chimeric antibody is a “class switched” antibody in which theclass or subclass has been changed from that of the parent antibody.Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region.

In some embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I., et al., Nature 332 (1988)323-329; Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V., et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); Osbourn, J.et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83(2000) 252-260 (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J., et al., J. Immunol. 151 (1993)2296-2308; framework regions derived from the consensus sequence ofhuman antibodies of a particular subgroup of light or heavy chainvariable regions (see, e.g., Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Presta, L. G., et al., J. Immunol. 151(1993) 2623-2632); human mature (somatically mutated) framework regionsor human germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

3. Human Antibodies

In certain embodiments, a dimeric polypeptide as reported herein isderived from a human antibody. Human antibodies can be produced usingvarious techniques known in the art. Human antibodies are describedgenerally in van Dijk, M. A. and van de Winkel, J. G., Curr. Opin.Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20(2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VELoCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immuno1.133 (1984) 3001-3005; Brodeur, B. R., et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boemer, P., et al., J. Immunol. 147(1991) 86-95). Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J., et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

4. Library-Derived Antibodies

In certain embodiments a heterodimeric polypeptide as reported herein iscomprised in a library-derived antibody. Library-derived antibodies maybe isolated by screening combinatorial libraries for antibodies with thedesired activity or activities.

For example, a variety of methods are known in the art for generatingphage display libraries and screening such libraries for antibodiespossessing the desired binding characteristics. Such methods arereviewed, e.g., in Hoogenboom, H. R. et al., Methods in MolecularBiology 178 (2001) 1-37 and further described, e.g., in the McCafferty,J. et al., Nature348 (1990) 552-554; Clackson, T. et al., Nature 352(1991) 624-628; Marks, J. D. et al., J. Mol. Biol. 222 (1992) 581-597;Marks, J. D. and Bradbury, A., Methods in Molecular Biology 248 (2003)161-175; Sidhu, S. S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C.V. et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc.Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, C. V. et al., J.Immunol. Methods 284 (2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G., et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self-antigens without any immunization as described by Griffiths,A. D., et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and US2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

5. Multispecific Antibodies

In certain embodiments, a heterodimeric polypeptide as reported hereinis comprised in a multispecific antibody, e.g. a bispecific antibody.Multispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different sites. In certain embodiments,one of the binding specificities is for a first antigen and the other isfor a different second antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of the same antigen.Bispecific antibodies may also be used to localize cytotoxic agents tocells, which express at least one of the antigens. Bispecific antibodiescan be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A., et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (scFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tuft, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” (see, US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

6. Antibody Variants

In certain embodiments, a heterodimeric polypeptide as reported hereinis comprised in an antibody. In further embodiment amino acid sequencevariants of the antibodies provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody. Amino acid sequencevariants of an antibody may be prepared by introducing appropriatemodifications into the nucleotide sequence encoding the antibody, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the antibody.

Any combination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in the Table below under the heading of “preferred substitutions”.More substantial changes are provided in the following Table under theheading of “exemplary substitutions”, and as further described below inreference to amino acid side chain classes. Amino acid substitutions maybe introduced into an antibody of interest and the products screened fora desired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu norleucine Leu (L) norleucine;Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity-matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contactantigen, with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described, e.g., in Hoogenboom, H. R. etal. in Methods in Molecular Biology 178 (2002) 1-37. In some embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding may be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen can be used.Such contact residues and neighboring residues may be targeted oreliminated as candidates for substitution. Variants may be screened todetermine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide, which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc-region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of theFc-region. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32. The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to anFc-region. For example, the amount of fucose in such antibody may befrom 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc-region (EUnumbering of Fc-region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J., et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N., et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda,Y., et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc-regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc-region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fe-Region Variants

In certain embodiments, one or more further amino acid modifications maybe introduced into a heterodimeric polypeptide as reported herein,thereby generating an Fc-region variant. The Fc-region variant may bederived from a human Fc-region sequence (e.g., a human IgG1, IgG2, IgG3or IgG4 Fc-region) comprising an amino acid modification (e.g. asubstitution/mutation) at one or more amino acid positions.

In certain embodiments, the invention contemplates a heterodimericpolypeptide that possesses some but not all effector functions, whichmake it a desirable candidate for applications in which the half-life ofthe dimeric polypeptide in vivo is important yet certain effectorfunctions (such as CDC and ADCC) are unnecessary or deleterious. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theheterodimeric polypeptide antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγR1, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492. Non-limitingexamples of in vitro assays to assess ADCC activity of a molecule ofinterest are described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom,I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; andHellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166(1987) 1351-1361). Alternatively, non-radioactive assays methods may beemployed (see, for example, ACTI™ non-radioactive cytotoxicity assay forflow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes, R.et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assaysmay also be carried out to confirm that the dimeric polypeptide isunable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3cbinding ELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171;Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M. S. and M.J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivoclearance/half-life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int. Immunol. 18(2006) 1759-1769).

Heterodimeric polypeptides with reduced effector function include thosewith substitution of one or more of Fc-region residues 238, 265, 269,270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc-region variantsinclude Fc-regions with substitutions at two or more of amino acidpositions 265, 269, 270, 297 and 327, including the so-called “DANA”Fc-region mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, a heterodimeric polypeptide variant comprises anFc-region with one or more amino acid substitutions, which improve ADCC,e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region(EU numbering of residues).

In some embodiments, alterations are made in the Fc-region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc-regionwith one or more substitutions therein, which improve binding of theFc-region to FcRn. Such Fc-region variants include those withsubstitutions at one or more of Fc-region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (U.S.Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc-region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered heterodimeric polypeptides, e.g., in analogy to “thioMAbs,”in which one or more residues of an antibody are substituted withcysteine residues. In particular embodiments, the substituted residuesoccur at accessible sites of the heterodimeric polypeptide. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the heterodimeric polypeptideand may be used to conjugate the heterodimeric polypeptide to othermoieties, such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and 5400 (EU numbering) of the heavy chainFc-region. Cysteine engineered dimeric polypeptides may be generated asdescribed, e.g., in U.S. Pat. No. 7,521,541.

e) Derivatives

In certain embodiments, a heterodimeric polypeptide as reported hereinmay be further modified to contain additional non-proteinaceous moietiesthat are known in the art and readily available. The moieties suitablefor derivatization of the heterodimeric polypeptide include but are notlimited to water-soluble polymers. Non-limiting examples of watersoluble polymers include, but are not limited to, polyethylene glycol(PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydridecopolymer, polyamino acids (either homopolymers or random copolymers),and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,pro-propylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched ornon-branched. The number of polymers attached to the dimeric polypeptidemay vary, and if more than one polymer is attached, they can be the sameor different molecules. In general, the number and/or type of polymersused for derivatization can be determined based on considerationsincluding, but not limited to, the particular properties or functions ofthe dimeric polypeptide to be improved, whether the dimeric polypeptidederivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of a heterodimeric polypeptide asreported herein and non-proteinaceous moiety that may be selectivelyheated by exposure to radiation are provided. In one embodiment, thenon-proteinaceous moiety is a carbon nanotube (Kam, N. W. et al., Proc.Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be ofany wavelength, and includes, but is not limited to, wavelengths that donot harm ordinary cells, but which heat the non-proteinaceous moiety toa temperature at which cells proximal to the dimericpolypeptide-non-proteinaceous moiety are killed.

f) Heterodimerization

There exist several approaches for CH3-modifications to enforce theheterodimerization, which are well described e.g. in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO2013157954, WO 2013096291. Typically in all such approaches the firstCH3 domain and the second CH3 domains are both engineered in acomplementary manner so that each CH3 domain (or the heavy chaincomprising it) cannot longer homodimerize with itself but is forced toheterodimerize with the complementary engineered other CH3 domain (sothat the first and second CH3 domain heterodimerize and no homodimersbetween the two first or the two second CH3 domains are formed). Thesedifferent approaches for improved heavy chain heterodimerization arecontemplated as different alternatives in combination with theheavy-light chain modifications (VH and VL exchange/replacement in onebinding arm and the introduction of substitutions of charged amino acidswith opposite charges in the CH1/CL interface) in the multispecificantibodies according to the invention which reduce light chainmispairing an Bence-Jones type side products.

In one preferred embodiment of the invention (in case the multispecificantibody comprises CH3 domains in the heavy chains) the CH3 domains ofsaid heterodimeric polypeptide according to the invention can be alteredby the “knob-into-holes” technology which is described in detail withseveral examples in e.g. WO 96/027011, Ridgway, J. B., et al., ProteinEng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16(1998) 677-681; WO 98/050431. In this method the interaction surfaces ofthe two CH3 domains are altered to increase the heterodimerization ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole”. The introduction of a disulfide bridge further stabilizesthe heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) andincreases the yield.

Thus in one embodiment of the invention said heterodimeric polypeptide(comprises a CH3 domain in each heavy chain and) is furthercharacterized in that

-   -   the first CH3 domain of the first heavy chain of the antibody        under a) and the second CH3 domain of the second heavy chain of        the antibody under b) each meet at an interface, which comprises        an original interface between the antibody CH3 domains.        -   wherein said interface is altered to promote the formation            of the multispecific antibody, wherein the alteration is            characterized in that:        -   i) the CH3 domain of one heavy chain is altered, so that            within the original interface of the CH3 domain of one heavy            chain that meets the original interface of the CH3 domain of            the other heavy chain within the multispecific antibody, an            amino acid residue is replaced with an amino acid residue            having a larger side chain volume, thereby generating a            protuberance within the interface of the CH3 domain of one            heavy chain, which is positionable in a cavity within the            interface of the CH3 domain of the other heavy chain and        -   ii) the CH3 domain of the other heavy chain is altered, so            that within the original interface of the second CH3 domain            that meets the original interface of the first CH3 domain            within the multispecific antibody        -   an amino acid residue is replaced with an amino acid residue            having a smaller side chain volume, thereby generating a            cavity within the interface of the second CH3 domain within            which a protuberance within the interface of the first CH3            domain is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one preferred embodiment, said heterodimeric polypeptide comprises anamino acid T366W mutation in the first CH3 domain of the “knobs-chain”and amino acid T366S, L368A, Y407V mutations in the second CH3 domain ofthe “hole-chain”. An additional interchain disulfide bridge between theCH3 domains can also be used (Merchant, A. M., et al., Nature Biotech.16 (1998) 677-681) e.g. by introducing an amino acid Y349C mutation intothe CH3 domain of the “hole-chain” and an amino acid E356C mutation oran amino acid S354C mutation into the CH3 domain of the “knobs-chain”.

In one preferred embodiment, said heterodimeric polypeptide (whichcomprises a CH3 domain in each heavy chain) comprises amino acid S354C,T366W mutations in one of the two CH3 domains and amino acid Y349C,T366S, L368A, Y407V mutations in the other of the two CH3 domains (theadditional amino acid S354C mutation in one CH3 domain and theadditional amino acid Y349C mutation in the other CH3 domain forming aninterchain disulfide bridge) (numbering according to Kabat).

Other techniques for CH3-modifications to enforcing theheterodimerization are contemplated as alternatives of the invention anddescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment the heterodimerization approach described in EP 1 870459A1, can be used alternatively. This approach is based on the by theintroduction of substitutions/mutations of charged amino acids with theopposite charge at specific amino acid positions of the in the CH3/CH3domain interface between both heavy chains. One preferred embodiment forsaid heterodimeric polypeptide are amino acid R409D; K370E mutations inthe first CH3 domain of the multispecific antibody and amino acid D399K;E357K mutations in the seconds CH3 domain of the multispecific antibody(numbering according to Kabat).

In another embodiment said heterodimeric polypeptide comprises a aminoacid T366W mutation in the CH3 domain of the “knobs chain” and aminoacid T366S, L368A, Y407V mutations in the CH3 domain of the “hole-chain”and additionally amino acid R409D; K370E mutations in the CH3 domain ofthe “knobs-chain” and amino acid D399K; E357K mutations in the CH3domain of the “hole-chain”.

In another embodiment said heterodimeric polypeptide comprises aminoacid S354C, T366W mutations in one of the two CH3 domains and amino acidY349C, T366S, L368A, Y407V mutations in the other of the two CH3 domainsor said multispecific antibody comprises amino acid Y349C, T366Wmutations in one of the two CH3 domains and amino acid S354C, T366S,L368A, Y407V mutations in the other of the two CH3 domains andadditionally amino acid R409D; K370E mutations in the CH3 domain of the“knobs-chain” and amino acid D399K; E357K mutations in the CH3 domain ofthe “hole-chain”.

In one embodiment the heterodimerization approach described inWO2013/157953 can be used alternatively. In one embodiment a first CH3domain comprises amino acid T366K mutation and a second CH3 domainpolypeptide comprises amino acid L351D mutation. In a further embodimentthe first CH3 domain comprises further amino acid L351K mutation. In afurther embodiment the second CH3 domain comprises further amino acidmutation selected from Y349E, Y349D and L368E (preferably L368E).

In one embodiment the heterodimerization approach described inWO2012/058768 can be used alternatively. In one embodiment a first CH3domain comprises amino acid L351Y, Y407A mutations and a second CH3domain comprises amino acid T366A, K409F mutations. In a furtherembodiment the second CH3 domain comprises a further amino acid mutationat position T411, D399, 5400, F405, N390, or K392 e.g. selected from a)T411 N, T411 R, T411Q, T411 K, T411D, T411E or T411W, b) D399R, D399W,D399Y or D399K, c S400E, S400D, S400R, or S400K F4051, F405M, F405T,F405S, F405V or F405W N390R, N390K or N390D K392V, K392M, K392R, K392L,K392F or K392E. In a further embodiment a first CH3 domain comprisesamino acid L351Y, Y407A mutations and a second CH3 domain comprisesamino acid T366V, K409F mutations. In a further embodiment a first CH3domain comprises amino acid Y407A mutations and a second CH3 domaincomprises amino acid T366A, K409F mutations. In a further embodiment thesecond CH3 domain comprises a further amino acid K392E, T411E, D399R andS400R mutations.

In one embodiment the heterodimerization approach described inWO2011/143545 can be used alternatively e.g. with the amino acidmodification at a position selected from the group consisting of 368 and409.

In one embodiment the heterodimerization approach described inWO2011/090762, which also uses the knobs-into-holes technology describedabove, can be used alternatively. In one embodiment a first CH3 domaincomprises amino acid T366W mutations and a second CH3 domain comprisesamino acid Y407A mutations. In one embodiment a first CH3 domaincomprises amino acid T366Y mutations and a second CH3 domain comprisesamino acid Y407T mutations.

In one embodiment the multispecific antibody is of IgG2 isotype and theheterodimerization approach described in WO2010/129304 can be usedalternatively.

In one embodiment the heterodimerization approach described inWO2009/089004 can be used alternatively. In one embodiment a first CH3domain comprises amino acid substitution of K392 or N392 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D), preferably K392D or N392D) and a second CH3 domain comprises aminoacid substitution of D399, E356, D356, or E357 with a positive-chargedamino acid (e.g. Lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K and more preferably D399K and E356K. In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negative-charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D)).

In one embodiment the heterodimerization approach described inWO2007/147901 can be used alternatively. In one embodiment a first CH3domain comprises amino acid K253E, D282K, and K322D mutations and asecond CH3 domain comprises amino acid D239K, E240K, and K292Dmutations.

In one embodiment the heterodimerization approach described inWO2007/110205 can be used alternatively.

E. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid(s) encoding a heterodimeric polypeptide asreported herein is(are) provided. Such nucleic acid may encode an aminoacid sequence comprising the first polypeptide and/or an amino acidsequence comprising the second polypeptide of the heterodimericpolypeptide. In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the first polypeptide of theheterodimeric polypeptide and an amino acid sequence comprising thesecond polypeptide of the heterodimeric polypeptide, or (2) a firstvector comprising a nucleic acid that encodes an amino acid sequencecomprising the first polypeptide of the heterodimeric polypeptide and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the second polypeptide of the heterodimericpolypeptide. In one embodiment, the host cell is eukaryotic, e.g. aChinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20cell). In one embodiment, a method of making a heterodimeric polypeptideas reported herein is provided, wherein the method comprises culturing ahost cell comprising a nucleic acid encoding the heterodimericpolypeptide, as provided above, under conditions suitable for expressionof the heterodimeric polypeptide, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

For recombinant production of a heterodimeric polypeptide as reportedherein, nucleic acid encoding a heterodimeric polypeptide, e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the variant Fc-region polypeptide(s) and the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of heterodimericpolypeptide-encoding vectors include prokaryotic or eukaryotic cellsdescribed herein. For example, heterodimeric polypeptides may beproduced in bacteria, in particular when glycosylation and Fc effectorfunction are not needed. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199, and 5,840,523. (See also Charlton, K. A., In: Methods inMolecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa,N.J. (2003), pp. 245-254, describing expression of antibody fragments inE. coli.). After expression, the heterodimeric polypeptide may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forheterodimeric polypeptide-encoding vectors, including fungi and yeaststrains whose glycosylation pathways have been “humanized” resulting inthe production of a dimeric polypeptide with a partially or fully humanglycosylation pattern. See Gemgross, T. U., Nat. Biotech. 22 (2004)1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated heterodimericpolypeptide are also derived from multicellular organisms (invertebratesand vertebrates). Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains have been identified whichmay be used in conjunction with insect cells, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (HEK293 or 293cells as described, e.g., in Graham, F. L., et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P., et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G., et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

F. Combination Treatment

In certain embodiments the heterodimeric polypeptide as reported hereinor pharmaceutical formulation as reported herein is administered alone(without an additional therapeutic agent) for the treatment of one ormore ocular vascular diseases described herein.

In other embodiments the heterodimeric polypeptide antibody orpharmaceutical formulation as reported herein is administered incombination with one or more additional therapeutic agents or methodsfor the treatment of one or more vascular eye diseases described herein.

In other embodiments, the heterodimeric polypeptide or pharmaceuticalformulation as reported herein is formulated in combination with one ormore additional therapeutic agents and administered for the treatment ofone or more vascular eye diseases described herein.

In certain embodiments, the combination treatments provided hereininclude that the heterodimeric polypeptide or pharmaceutical formulationas reported herein is administered sequentially with one or moreadditional therapeutic agents for the treatment of one or more ocularvascular diseases described herein.

The additional therapeutic agents include, but are not limited to,Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (anti-VEGF PEGylatedaptamer), squalamine, RETAANE™ (anecortave acetate for depot suspension;Alcon, Inc.), Combretastatin A4 Prodrug (CA4P), MACUGEN™, MIFEPREX™(mifepristone-ru486), subtenon triamcinolone acetonide, intravitrealcrystalline triamcinolone acetonide, Prinomastat (AG3340-syntheticmatrix metalloproteinase inhibitor, Pfizer), fluocinolone acetonide(including fluocinolone intraocular implant, Bausch & Lomb/ControlDelivery Systems), VEGFR inhibitors (Sugen), VEGF-Trap(Regeneron/Aventis), VEGF receptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787) and SU1 1248 (sunitinib), linomide, andinhibitors of integrin v.beta.3 function and angiostatin.

Other pharmaceutical therapies that can be used in combination with theheterodimeric polypeptide or pharmaceutical formulation as reportedherein, including, but are not limited to, VISUDYNE™ with use of anon-thermal laser, PKC 412, Endovion (NeuroSearch A/S), neurotrophicfactors, including by way of example Glial Derived Neurotrophic Factorand Ciliary Neurotrophic Factor, diatazem, dorzolamide, Phototrop,9-cis-retinal, eye medication (including Echo Therapy) includingphospholine iodide or echothiophate or carbonic anhydrase inhibitors,AE-941 (AEtema Laboratories, Inc.), Sima-027 (Sima Therapeutics, Inc.),pegaptanib (NeXstar Pharmaceuticals/Gilead Sciences), neurotrophins(including, by way of example only, NT-4/5, Genentech), CandS (AcuityPharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrinantagonists (including those from Jerini AG and Abbott Laboratories),EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (asused, for example, by EntreMed, Inc.), cardiotrophin-1 (Genentech),2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries),NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan),LYN-002 (Lynkeus Biotech), microalgal compound (Aquasearch/Albany, MeraPharmaceuticals), D-9120 (Celltech Group plc.), ATX-S10 (HamamatsuPhotonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors(Allergan, SUGEN, Pfizer), NX-278-L (NeXstar Pharmaceuticals/GileadSciences), Opt-24 (OPTIS France SA), retinal cell ganglionneuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives(Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals),cyclosporine A, timited retinal translocation, photodynamic therapy,(including, by way of example only, receptor-targeted PDT, Bristol-MyersSquibb, Co.; porfimer sodium for injection with PDT; verteporfin, QLTInc.; rostaporfin with PDT, Miravent Medical Technologies; talaporfinsodium with PDT, Nippon Petroleum; motexafin lutetium, Pharmacyclics,Inc.), antisense oligonucleotides (including, by way of example,products tested by Novagali Pharma SA and ISIS-13650, IsisPharmaceuticals), laser photocoagulation, drusen lasering, macular holesurgery, macular translocation surgery, implantable miniaturetelescopes, Phi-Motion Angiography (also known as Micro-Laser Therapyand Feeder Vessel Treatment), Proton Beam Therapy, microstimulationtherapy, Retinal Detachment and Vitreous Surgery, Scleral Buckle,Submacular Surgery, Transpupillary Thermotherapy, Photosystem I therapy,use of RNA interference (RNAi), extracorporeal rheopheresis (also knownas membrane differential filtration and Rheotherapy), microchipimplantation, stem cell therapy, gene replacement therapy, ribozyme genetherapy (including gene therapy for hypoxia response element, OxfordBiomedica; Lentipak, Genetix; PDEF gene therapy, GenVec),photoreceptor/retinal cells transplantation (including transplantableretinal epithelial cells, Diacrin, Inc.; retinal cell transplant, CellGenesys, Inc.), and acupuncture.

Any anti-angiogenic agent can be used in combination with theheterodimeric polypeptide or pharmaceutical formulation as reportedherein, including, but not limited to, those listed by Carmeliet andJain (Nature 407 (2000) 249-257). In certain embodiments, theanti-angiogenic agent is another VEGF antagonist or a VEGF receptorantagonist such as VEGF variants, soluble VEGF receptor fragments,aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFRantibodies, low molecule weight inhibitors of VEGFR tyrosine kinases andany combinations thereof and these include anti-VEGF aptamers (e.g.Pegaptanib), soluble recombinant decoy receptors (e.g. VEGF Trap). Incertain embodiments, the anti-angiogenic agent is includecorticosteroids, angiostatic steroids, anecortave acetate, angiostatin,endostatin, small interfering RNA's decreasing expression of VEGFR orVEGF ligand, post-VEGFR blockade with tyrosine kinase inhibitors, MMPinhibitors, IGFBP3, SDF-1 blockers, PEDF, gamma-secretase, Delta-likeligand 4, integrin antagonists, HIF-1 alpha blockade, protein kinase CK2blockade, and inhibition of stem cell (i.e. endothelial progenitor cell)homing to the site of neovascularization using vascular endothelialcadherin (CD-144) and stromal derived factor (SDF)-I antibodies. Smallmolecule RTK inhibitors targeting VEGF receptors including PTK787 canalso be used. Agents that have activity against neovascularization thatare not necessarily anti-VEGF compounds can also be used and includeanti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolismus,temsirolismus, cyclospohne, anti-TNF agents, anti-complement agents, andnon-steroidal anti-inflammatory agents. Agents that are neuroprotectiveand can potentially reduce the progression of dry macular degenerationcan also be used, such as the class of drugs called the ,neurosteroids“.These include drugs such as dehydroepiandrosterone (DHEA) (Brand names:Prastera(R) and Fidelin(R)), dehydroepiandrosterone sulfate, andpregnenolone sulfate. Any AMD (age-related macular degeneration)therapeutic agent can be used in combination with the dimericpolypeptide or pharmaceutical formulation as reported herein, includingbut not limited to verteporfin in combination with PDT, pegaptanibsodium, zinc, or an antioxidant(s), alone or in any combination.

G. Pharmaceutical Formulations

Pharmaceutical formulations of a heterodimeric polypeptide as reportedherein are prepared by mixing such heterodimeric polypeptide having thedesired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences, 16th edition,Osol, A. (ed.) (1980)), in the form of lyophilized formulations oraqueous solutions. Pharmaceutically acceptable carriers are generallynontoxic to recipients at the dosages and concentrations employed, andinclude, but are not limited to: buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyl dimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone);amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US 2005/0260186 and US2006/0104968. In one aspect, a sHASEGP is combined with one or moreadditional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethyl cellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

H. Therapeutic Methods and Compositions

Any of the heterodimeric polypeptides as reported herein may be used intherapeutic methods.

In one aspect, a heterodimeric polypeptide as reported herein for use asa medicament is provided. In further aspects, a heterodimericpolypeptide for use in treating ocular vascular diseases is provided. Incertain embodiments, a heterodimeric polypeptide for use in a method oftreatment is provided. In certain embodiments, the invention provides aheterodimeric polypeptide for use in a method of treating an individualhaving an ocular vascular disease comprising administering to theindividual an effective amount of the heterodimeric polypeptide asreported herein. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described above in section D. Infurther embodiments, the invention provides a heterodimeric polypeptidefor use in inhibiting angiogenesis in the eye. In certain embodiments,the invention provides a heterodimeric polypeptide for use in a methodof inhibiting angiogenesis in an individual comprising administering tothe individual an effective of the heterodimeric polypeptide to inhibitangiogenesis. An “individual” according to any of the above embodimentsis in one preferred embodiment a human.

In a further aspect, the invention provides for the use of aheterodimeric polypeptide in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment of anocular vascular disease. In a further embodiment, the medicament is foruse in a method of treating an ocular vascular disease comprisingadministering to an individual having an ocular vascular disease aneffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, e.g., as described above. Ina further embodiment, the medicament is for inhibiting angiogenesis. Ina further embodiment, the medicament is for use in a method ofinhibiting angiogenesis in an individual comprising administering to theindividual an amount effective of the medicament to inhibitangiogenesis. An “individual” according to any of the above embodimentsmay be a human.

In a further aspect, the invention provides a method for treating avascular eye disease. In one embodiment, the method comprisesadministering to an individual having such a vascular eye disease aneffective amount of a heterodimeric polypeptide as reported herein. Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for inhibitingangiogenesis in the eye in an individual. In one embodiment, the methodcomprises administering to the individual an effective amount of aheterodimeric polypeptide as reported herein to inhibit angiogenesis. Inone embodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the heterodimeric polypeptides as reported herein,e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of theheterodimeric polypeptides as reported herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the heterodimeric polypeptides as reported herein andat least one additional therapeutic agent, e.g., as described below.

Heterodimeric polypeptide as reported herein can be used either alone orin combination with other agents in a therapy. For instance, aheterodimeric polypeptide as reported herein may be co-administered withat least one additional therapeutic agent

A heterodimeric polypeptide as reported herein (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Heterodimeric polypeptides as reported herein would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The heterodimeric polypeptide need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of heterodimeric polypeptidepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of aheterodimeric polypeptide as reported herein (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of heterodimericpolypeptide, the severity and course of the disease, whether theheterodimeric polypeptide is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the heterodimeric polypeptide, and the discretion of the attendingphysician. The heterodimeric polypeptide is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5mg/kg-10 mg/kg) of heterodimeric polypeptide can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentwould generally be sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the heterodimeric polypeptidewould be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, oneor more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (orany combination thereof) may be administered to the patient. Such dosesmay be administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the dimeric polypeptide). An initial higherloading dose, followed by one or more lower doses may be administered.The progress of this therapy is easily monitored by conventionaltechniques and assays.

III. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition, which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a heterodimeric polypeptide as reported herein. The labelor package insert indicates that the composition is used for treatingthe condition of choice. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises a heterodimeric polypeptide asreported herein; and (b) a second container with a composition containedtherein, wherein the composition comprises a further otherwisetherapeutic agent. The article of manufacture in this embodiment of theinvention may further comprise a package insert indicating that thecompositions can be used to treat a particular condition. Alternatively,or additionally, the article of manufacture may further comprise asecond (or third) container comprising a pharmaceutically acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate as reported herein in place of or in additionto a heterodimeric polypeptide as reported herein.

IV. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Methods

Electrospray ionization mass spectrometry (ESI-MS)

Protein aliquots (50 μg) are deglycosylated by adding 0.5 μL N-Glycanaseplus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) to obtain afinal sample volume of 115 μL. The mixture is incubated at 37° C. for 18h. Afterwards for reduction and denaturing 60 μL 0.5 M TCEP (Pierce) in4 M guanidine*HCl (Pierce) and 50 μL 8 M guanidine*HCl are added. Themixture is incubated at 37° C. for 30 min. Samples are desalted by sizeexclusion chromatography (Sepharose G-25, isocratic, 40% acetonitrilewith 2% formic acid). ESI mass spectra (+ve) are recorded on a Q-TOFinstrument (maXis, Bruker) equipped with a nano ESI source (TriVersaNanoMate, Advion). MS parameter settings are as follows: Transfer:Funnel RF, 400 Vpp; ISCID Energy, 0 eV; Multipole RF, 400 Vpp;Quadrupole: Ion Energy, 4.0 eV; Low Mass, 600 m/z; Source: Dry Gas, 8L/min; Dry Gas Temperature, 160° C.; Collision Cell: Collision Energy,10 eV; Collision RF: 2000 Vpp; Ion Cooler: Ion Cooler RF, 300 Vpp;Transfer Time: 120 μs; Pre Puls Storage, 10 μs; scan range m/z 600 to2000. For data evaluation in-house developed software (MassAnalyzer) isused.

FcRn surface plasmon resonance (SPR) analysis

The binding properties of wild-type antibody and the mutants to FcRn areanalyzed by surface plasmon resonance (SPR) technology using a BIAcoreT100 instrument (BIAcore AB, Uppsala, Sweden). This system is wellestablished for the study of molecular interactions. It allows acontinuous real-time monitoring of ligand/analyte bindings and thus thedetermination of kinetic parameters in various assay settings.SPR-technology is based on the measurement of the refractive index closeto the surface of a gold-coated biosensor chip. Changes in therefractive index indicate mass changes on the surface caused by theinteraction of immobilized ligand with analyte injected in solution. Ifmolecules bind to an immobilized ligand on the surface the massincreases, in case of dissociation the mass decreases. In the currentassay, the FcRn receptor is immobilized onto a BIAcore CM5-biosensorchip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to alevel of 400 Response units (RU). The assay is carried out at roomtemperature with PBS, 0.05% Tween20 pH 6.0 (GE Healthcare Bioscience) asrunning and dilution buffer. 200 nM of samples are injected at a flowrate of 50 μL/min at room temperature. Association time is 180 sec.,dissociation phase took 360 sec. Regeneration of the chip surface isreached by a short injection of HBS-P, pH 8.0. Evaluation of SPR-data isperformed by comparison of the biological response signal height at 180sec. after injection and at 300 sec. after injection. The correspondingparameters are the RU max level (180 sec. after injection) and latestability (300 sec. after end of injection).

Protein A surface plasmon resonance (SPR) analysis

The assay is based on surface plasmon resonance spectroscopy. Protein Ais immobilized onto the surface of a SPR biosensor. By injecting thesample into the flow cells of the SPR spectrometer it forms a complexwith the immobilized protein A resulting in an increasing mass on thesensor chip surface, and therefore to a higher response (as 1 RU isdefined as 1 pg/mm²). Afterwards the sensor chip is regenerated bydissolving the sample-protein A-complex. The gained responses are thenevaluated for the signal high in response units (RU) and thedissociation behavior.

Around 3500 response units (RU) of protein A (20 μg/mL) are coupled ontoa CM5 chip (GE Healthcare) at pH 4.0 by using the amine coupling kit ofGE Healthcare.

The sample and system buffer is HBS-P+(0.01 M HEPES, 0.15 M NaCl, 0.005%Surfactant P20 Sterile-filtered, pH 7.4). Flow cell temperature is setto 25° C. and sample compartment temperature to 12° C. The system isprimed with running buffer. Then, a 5 nM solutions of the sampleconstructs are injected for 120 seconds with a flow rate of 30 μL/min,followed by a 300 seconds dissociation phase. Then the sensor chipsurface is regenerated by two 30 seconds long injections of Glycine-HClpH 1.5 at a flow rate of 30 μL/min. Each sample is measured as atriplicate.

The term “with (the) mutation IHH-AAA” as used herein refers thecombination of the mutations I253A (Ile253A1a), H310A (His310A1a), andH435A (His435A1a) in a constant heavy chain region of IgG1 or IgG4subclass (numbering according to the Kabat EU index numbering system),the term “with (the) mutation HHY-AAA” as used herein refers thecombination of the mutations H310A (His310A1a), H433A (His433A1a) andY436A (Tyr436A1a) in a constant heavy chain region of IgG1 or IgG4subclass (numbering according to the Kabat EU index numbering system),the term “with (the) mutation P329G LALA” as used herein refers to thecombination of the mutations L234A (Leu234A1a), L235A (Leu235A1a) andP329G (Pro329Gly) in a constant heavy chain region of IgG1 subclass(numbering according to the Kabat EU index numbering system), and theterm “with (the) mutation SPLE” as used herein refers to the combinationof the mutations S228P (Ser228Pro) and L235E (Leu235Glu) in a constantheavy chain region of IgG4 subclass (numbering according to the Kabat EUindex numbering system).

General

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Amino acidresidues of antibody chains are numbered and referred to according to EUnumbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969)78-85; Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

Recombinant DNA Techniques

Standard methods are used to manipulate DNA as described in Sambrook, J.et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents are used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments are ordered according to given specifications atGeneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences are determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH(Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 is usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies expression vectors fortransient expression (e.g. in HEK293-F cells) based either on a cDNAorganization with or without a CMV-Intron A promoter or on a genomicorganization with a CMV promoter are used.

Beside the antibody expression cassette the vectors contains:

-   -   an origin of replication which allows replication of this vector        in E. coli,    -   a β-lactamase gene which confers ampicillin resistance in E.        coli, and    -   the dihydrofolate reductase gene from Mus musculus as a        selectable marker in eukaryotic cells.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end,    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   in the case of the cDNA organization followed by the Intron A        sequence,    -   a 5′-untranslated region of a human immunoglobulin gene,    -   a nucleic acid encoding an immunoglobulin heavy chain signal        sequence,    -   a nucleic acid encoding the human antibody chain (wild-type or        with domain exchange) either as cDNA or in genomic organization        with the immunoglobulin exon-intron organization,    -   a 3′ non-translated region with a polyadenylation signal        sequence, and    -   unique restriction site(s) at the 3′ end.

The nucleic acids encoding the antibody chains are generated by PCRand/or gene synthesis and assembled by known recombinant methods andtechniques by connection of the according nucleic acid segments e.g.using unique restriction sites in the respective vectors. The subclonednucleic acid sequences are verified by DNA sequencing. For transienttransfections larger quantities of the vectors are prepared by vectorpreparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques are used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

The bispecific antibodies are expressed by transient co-transfection ofthe respective expression vectors in HEK29-F cells growing in suspensionas described below.

Example 1 Expression and Purification

Transient Transfections in HEK293-F System

The monospecific and bispecific antibodies are generated by transienttransfection with the respective vectors (e.g. encoding the heavy andmodified heavy chain, as well as the corresponding light and modifiedlight chain) using the HEK293-F system (Invitrogen) according to themanufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growingin suspension either in a shake flask or in a stirred fermenter inserum-free FreeStyle™ 293 expression medium (Invitrogen) are transfectedwith a mix of the respective expression vectors and 293fectin™ or fectin(Invitrogen). For 2 L shake flask (Corning) HEK293-F cells are seeded ata density of 1*10⁶ cells/mL in 600 mL and incubated at 120 rpm, 8% CO₂.The day after the cells are transfected at a cell density of approx.1.5*10⁶ cells/mL with approx. 42 mL mix of A) 20 mL Opti-MEM(Invitrogen) with 600 μg total vector DNA (1 μg/mL) encoding the heavyor modified heavy chain, respectively and the corresponding light chainin an equimolar ratio and B) 20 ml Opti-MEM with 1.2 mL 293 fectin orfectin (2 μL/mL). According to the glucose consumption glucose solutionis added during the course of the fermentation. The supernatantcontaining the secreted antibody is harvested after 5-10 days andantibodies are either directly purified from the supernatant or thesupernatant is frozen and stored.

Purification

Bispecific antibodies are purified from cell culture supernatants byaffinity chromatography using MabSelectSure-Sepharose™, hydrophobicinteraction chromatography using butyl-Sepharose (GE Healthcare, Sweden)and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.

Briefly, sterile filtered cell culture supernatants are captured on aMabSelectSure resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mMKH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibrationbuffer and eluted with 25 mM sodium citrate at pH 3.0. The elutedantibody fractions are pooled and neutralized with 2 M Tris, pH 9.0. Theantibody pools are prepared for hydrophobic interaction chromatographyby adding 1.6 M ammonium sulfate solution to a final concentration of0.8 M ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid.After equilibration of the butyl-Sepharose resin with 35 mM sodiumacetate, 0.8 M ammonium sulfate, pH 5.0, the antibodies are applied tothe resin, washed with equilibration buffer and eluted with a lineargradient to 35 mM sodium acetate pH 5.0. The (monospecific orbispecific) antibody containing fractions were pooled and furtherpurified by size exclusion chromatography using a Superdex 200 26/60 GL(GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mMNaCl, pH 6.0. The (monospecific or bispecific) antibody containingfractions were pooled, concentrated to the required concentration usingVivaspin ultrafiltration devices (Sartorius Stedim Biotech S. A.,France) and stored at −80° C.

Purity and antibody integrity can be analyzed after each purificationstep by CE-SDS using microfluidic Labchip technology (Caliper LifeScience, USA). Five μL of protein solution is prepared for CE-SDSanalysis using the HT Protein Express Reagent Kit accordingmanufacturer's instructions and analyzed on Labchip GXII system using aHT Protein Express Chip. Data are analyzed using Labchip GX Software.

The aggregate content of antibody samples can be analyzed byhigh-performance SEC using a Superdex 200 analytical size-exclusioncolumn (GE Healthcare, Sweden) in 2xPBS (20 mM Na₂HPO₄, 2 mM KH₂PO₄, 274mM NaCl and 5.4 mM KCl, pH 7.4) running buffer at 25° C. 25 pg proteinare injected on the column at a flow rate of 0.75 mL/min and elutedisocratic over 50 minutes.

Example 2 FeRn Chromatography

Coupling to Streptavidin Sepharose:

One gram streptavidin Sepharose (GE Healthcare) is added to thebiotinylated and dialyzed receptor and incubated for two hours withshaking. The receptor derivatized Sepharose is filled in a 1 mL XKcolumn (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

Conditions:

column dimensions: 50 mm×5 mm

bed height: 5 cm

loading: 50 μg sample

equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5

elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted to pH 8.8

elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer,10 CV elution buffer

1-15. (canceled)
 16. A method for treating an ocular vascular disease, the method comprising administering to a subject in need thereof a composition comprising an effective amount of a heterodimeric polypeptide to transport a soluble receptor ligand from the eye to the blood, wherein the heterodimeric polypeptide specifically binds the ligand and wherein the heterodimeric polypeptide comprises: a first polypeptide comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, and a second polypeptide comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V (hole-chain) and the second polypeptide comprises the mutations S354C and T366W (knob-chain), and wherein the first polypeptide (hole-chain) comprises one of the mutations selected from each of i) and ii), wherein i) and ii) are: (i) I253A or I253G, and (ii) L314A or L314G or L314D, and wherein the first polypeptide and the second polypeptide are connected by one or more disulfide bridges, and wherein the CH3-domain of the first polypeptide and the CH3-domain of the second polypeptide both bind or both do not bind to protein A, and wherein the knob-chain does not comprise I253A, I253G, L314A, L314G or L314D mutations (numbering according to the Kabat EU index).
 17. The method according to claim 16, wherein the heterodimeric polypeptide further comprises: a) the mutation T250Q, or b) the mutations T250Q and one of the mutations T256E and T256A, or c) one of the mutations T256E and T256A.
 18. The method according to claim 16, wherein the heterodimeric polypeptide further comprises: a) one of the mutations L251A, L251G, and L251D, or b) one of the mutations H310A and H310G.
 19. The method according to claim 16, wherein the heterodimeric polypeptide further comprises at least one mutation selected from one or more of i) to vi), wherein i) to vi) are: (i) T250Q, (ii) M252Y, (iii) S254T, (iv) T256E or T256A, (v) T307A or T307H or T307Q or T307P, and (vi) Q311H.
 20. The method according to claim 16, wherein the immunoglobulin hinge regions, the immunoglobulin CH2-domains, and the immunoglobulin CH3-domains of the first and second polypeptides are of the human IgG1 subclass.
 21. The method according to claim 16, wherein the first polypeptide and the second polypeptide further comprise the mutations L234A and L235A.
 22. The method according to claim 16, wherein the first polypeptide and the second polypeptide further comprise the mutation P329G.
 23. The method according to claim 16, wherein the immunoglobulin hinge regions, the immunoglobulin CH2-domains, and the immunoglobulin CH3-domains of the first and second polypeptides are of the human IgG4 subclass.
 24. The method according to claim 16, wherein the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.
 25. The method according to claim 16, wherein the first polypeptide and the second polypeptide further comprise the mutation P329G.
 26. The method according to claim 16, wherein the heterodimeric polypeptide is a full-length bispecific antibody.
 27. The method according to claim 18, wherein the heterodimeric polypeptide further comprises: a) the mutation T250Q, or b) the mutation T250Q and one of the mutations T256E and T256A, or c) one of the mutations T256E and T256A.
 28. The method according to claim 18, wherein the heterodimeric polypeptide further comprises: a) one of the mutations L251A, L251G, and L251D, and b) one of the mutations H310A and H310G.
 29. The method according to claim 27, wherein the heterodimeric polypeptide further comprises: a) one of the mutations L251A, L251G, and L251D, and b) one of the mutations H310A and H310G.
 30. The method according to claim 27, wherein the heterodimeric polypeptide further comprises: (i) a mutation selected from T307A, T307H, T307Q, and T307P, or (ii) one or more of the mutations selected from the group consisting of Q311H, M252Y, and S254T.
 31. The method according to claim 30, wherein the heterodimeric polypeptide further comprises: (i) a mutation selected from T307A, T307H, T307Q, and T307P, and (ii) one or more of the mutations selected from the group consisting of Q311H, M252Y, and S254T.
 32. The method according to claim 16, wherein the composition is administered by topical application to the cornea or by intravitreal application.
 33. The method according to claim 16, wherein the ocular vascular disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD), diabetic macular edema (DME), cystoid macular edema (CME), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoid macular edema, vasculitis (e.g. central retinal vein occlusion), papilloedema, retinitis, conjunctivitis, uveitis, choroiditis, multifocal choroiditis, ocular histoplasmosis, blepharitis, and dry eye (Sjogren's disease).
 34. The method according to claim 16, further comprising administering one or more additional therapeutic agents for a vascular eye disease.
 35. The method according to claim 16, wherein the heterodimeric polypeptide acts to inhibit angiogenesis in the eye of said subject. 